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1.
Proc Natl Acad Sci U S A ; 120(24): e2210113120, 2023 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-37279279

RESUMEN

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Pulmón , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Madre/metabolismo , Diferenciación Celular , Linaje de la Célula , Organoides , Células Epiteliales/metabolismo
2.
Development ; 149(20)2022 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-36278875

RESUMEN

Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.


Asunto(s)
Esófago , Organoides , Adulto , Humanos , Células Madre , Células Epiteliales/metabolismo , Diferenciación Celular
3.
Am J Physiol Lung Cell Mol Physiol ; 324(4): L433-L444, 2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-36791060

RESUMEN

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.


Asunto(s)
Factores de Crecimiento de Fibroblastos , Células Madre Mesenquimatosas , Animales , Humanos , Ratones , Factores de Crecimiento de Fibroblastos/genética , Pulmón/metabolismo , Morfogénesis/fisiología , Organogénesis/genética
4.
Development ; 145(6)2018 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-29467240

RESUMEN

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.


Asunto(s)
Adenoma/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenoma/genética , Línea Celular Tumoral , Colon/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Mucosa Intestinal/citología , Organoides/metabolismo , Transducción de Señal
5.
Artículo en Inglés | MEDLINE | ID: mdl-32540978

RESUMEN

Toxoplasmosis is a potentially fatal infection for immunocompromised people and the developing fetus. Current medicines for toxoplasmosis have high rates of adverse effects that interfere with therapeutic and prophylactic regimens. Endochin-like quinolones (ELQs) are potent inhibitors of Toxoplasma gondii proliferation in vitro and in animal models of acute and latent infection. ELQ-316, in particular, was found to be effective orally against acute toxoplasmosis in mice and highly selective for T. gondii cytochrome b over human cytochrome b Despite its oral efficacy, the high crystallinity of ELQ-316 limits oral absorption, plasma concentrations, and therapeutic potential. A carbonate ester prodrug of ELQ-316, ELQ-334, was created to decrease crystallinity and increase oral bioavailability, which resulted in a 6-fold increase in both the maximum plasma concentration (Cmax) and the area under the curve (AUC) of ELQ-316. The increased bioavailability of ELQ-316, when administered as ELQ-334, resulted in efficacy against acute toxoplasmosis greater than that of an equivalent dose of ELQ-316 and had efficacy against latent toxoplasmosis similar to that of ELQ-316 administered intraperitoneally. Treatment with carbonate ester prodrugs is a successful strategy to overcome the limited oral bioavailability of ELQs for the treatment of toxoplasmosis.


Asunto(s)
Profármacos , Quinolonas , Toxoplasma , Toxoplasmosis Animal , Animales , Encéfalo/parasitología , Carbonatos , Ésteres , Ratones , Toxoplasmosis Animal/tratamiento farmacológico
6.
Respirology ; 24(11): 1073-1080, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-30845351

RESUMEN

BACKGROUND AND OBJECTIVE: In vivo evaluation of the microstructural differences between asthmatic and non-asthmatic airways and their functional consequences is relevant to understanding and, potentially, treating asthma. In this study, we use endobronchial optical coherence tomography to investigate how allergic airways with asthma differ from allergic non-asthmatic airways in baseline microstructure and in response to allergen challenge. METHODS: A total of 45 subjects completed the study, including 20 allergic, mildly asthmatic individuals, 22 non-asthmatic allergic controls and 3 healthy controls. A 3-cm airway segment in the right middle and right upper lobe were imaged in each subject immediately before and 24 h following segmental allergen challenge to the right middle lobe. Relationships between optical airway measurements (epithelial and mucosal thicknesses, mucosal buckling and mucus) and airway obstruction (FEV1 /FVC (forced expiratory volume in 1 s/forced vital capacity) and FEV1 % (FEV1 as a percentage of predictive value)) were investigated. RESULTS: Significant increases at baseline and in response to allergen were observed for all four of our imaging metrics in the asthmatic airways compared to the non-asthmatic airways. Epithelial thickness and mucosal buckling exhibited a significant relationship to FEV1 /FVC in the asthmatic group. CONCLUSION: Simultaneous assessments of airway microstructure, buckling and mucus revealed both structural and functional differences between the mildly asthmatic and control groups, with airway buckling seeming to be the most relevant factor. The results of this study demonstrate that a comprehensive, microstructural approach to assessing the airways may be important in future asthma studies as well as in the monitoring and treatment of asthma.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Alérgenos/inmunología , Asma , Pulmón , Hipersensibilidad Respiratoria , Tomografía de Coherencia Óptica/métodos , Adulto , Asma/diagnóstico , Asma/inmunología , Asma/fisiopatología , Pruebas de Provocación Bronquial/métodos , Broncoscopía/métodos , Femenino , Humanos , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Masculino , Pruebas de Función Respiratoria/métodos , Hipersensibilidad Respiratoria/diagnóstico , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/fisiopatología
7.
Physiology (Bethesda) ; 32(3): 246-260, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28404740

RESUMEN

The main function of the lung is to support gas exchange, and defects in lung development or diseases affecting the structure and function of the lung can have fatal consequences. Most of what we currently understand about human lung development and disease has come from animal models. However, animal models are not always fully able to recapitulate human lung development and disease, highlighting an area where in vitro models of the human lung can compliment animal models to further understanding of critical developmental and pathological mechanisms. This review will discuss current advances in generating in vitro human lung models using primary human tissue, cell lines, and human pluripotent stem cell derived lung tissue, and will discuss crucial next steps in the field.


Asunto(s)
Homeostasis , Técnicas In Vitro/métodos , Enfermedades Pulmonares/fisiopatología , Pulmón/crecimiento & desarrollo , Pulmón/fisiopatología , Modelos Biológicos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Modelos Animales de Enfermedad , Humanos , Técnicas In Vitro/tendencias , Enfermedades Pulmonares/metabolismo , Células Madre Pluripotentes/fisiología , Regeneración
8.
Tissue Eng Part A ; 28(21-22): 893-906, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36029210

RESUMEN

Human lung organoids (HLOs) are enabling the study of human lung development and disease by modeling native organ tissue structure, cellular composition, and cellular organization. In this report, we demonstrate that HLOs derived from human pluripotent stem cells cultured in alginate, a fully defined nonanimal product substrate, exhibit enhanced cellular differentiation compared with HLOs cultured in the commercially available Matrigel. More specifically, we observed an earlier onset and increase in the number of multiciliated cells, along with mucus producing MUC5AC+ goblet-like cells that were not observed in HLOs cultured in Matrigel. The epithelium in alginate-grown HLOs was organized in a pseudostratified epithelium with airway basal cells lining the basal lamina, but with the apical surface of cells on the exterior of the organoid. We further observed that HLOs cultured in Matrigel exhibited mesenchymal overgrowth that was not present in alginate cultures. The containment of the mesenchyme within HLOs in alginate enabled modeling of key features of idiopathic pulmonary fibrosis (IPF) by treatment with transforming growth factor ß (TGFß). TGFß treatment resulted in morphological changes including an increase in mesenchymal growth, increased expression of IPF markers, and decreased numbers of alveolar-like cells. This culture system provides a model to study the interaction of the mesenchyme with the epithelium during lung development and diseased states such as IPF.


Asunto(s)
Alginatos , Organoides , Humanos , Alginatos/farmacología , Diferenciación Celular , Pulmón , Factor de Crecimiento Transformador beta
9.
Dev Cell ; 57(13): 1598-1614.e8, 2022 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-35679862

RESUMEN

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.


Asunto(s)
Células Madre Mesenquimatosas , Organogénesis , Humanos , Pulmón , Organoides , Vía de Señalización Wnt
10.
iScience ; 23(5): 101083, 2020 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-32380423

RESUMEN

Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (<1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases.

11.
Dev Cell ; 53(1): 117-128.e6, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32109386

RESUMEN

Bud tip progenitor cells give rise to all murine lung epithelial lineages and have been described in the developing human lung; however, the mechanisms controlling human bud tip differentiation into specific lineages are unclear. Here, we used homogeneous human bud tip organoid cultures and identified SMAD signaling as a key regulator of the bud tip-to-airway transition. SMAD induction led to the differentiation of airway-like organoids possessing functional basal cells capable of clonal expansion and multilineage differentiation. To benchmark in vitro-derived organoids, we developed a single-cell mRNA sequencing atlas of the human lung from 11.5 to 21 weeks of development, which revealed high degrees of similarity between the in vitro-derived and in vivo airway. Together, this work sheds light on human airway differentiation in vitro and provides a single-cell atlas of the developing human lung.


Asunto(s)
Diferenciación Celular/fisiología , Células Epiteliales/citología , Organoides/citología , Células Madre Pluripotentes/citología , Humanos , Pulmón/citología , Ingeniería de Tejidos/métodos
12.
Nat Protoc ; 14(2): 518-540, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30664680

RESUMEN

The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-posterior and dorsal-ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral-anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids. The resulting human lung organoids possess cell types and structures that resemble the bronchi/bronchioles of the developing human airway surrounded by lung mesenchyme and cells expressing alveolar-cell markers. The bud tip progenitor organoids possess a population of highly proliferative multipotent cells with in vitro multilineage differentiation potential and in vivo engraftment potential. Human lung organoids can be generated from hPSCs in 50-85 d, and bud tip progenitor organoids can be generated in 22 d. The two hPSC-derived models presented here have been benchmarked with human fetal tissue and found to be representative of human fetal-like tissue. The bud tip progenitor organoids are thus ideal for exploring epithelial fate decisions, while the human lung organoids can be used to model epithelial-mesenchymal cross-talk during human lung development. In addition to their applications in developmental biology, human lung organoids and bud tip progenitor organoids may be implemented in regenerative medicine, tissue engineering, and pharmaceutical safety and efficacy testing.


Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales/citología , Organoides/citología , Células Madre Pluripotentes/citología , Mucosa Respiratoria/citología , Ingeniería de Tejidos/métodos , Benchmarking , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Células Epiteliales/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , Organogénesis , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Medicina Regenerativa/métodos , Mucosa Respiratoria/metabolismo
13.
JCI Insight ; 52019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30913038

RESUMEN

Many lung diseases result from a failure of efficient regeneration of damaged alveolar epithelial cells (AECs) after lung injury. During regeneration, AEC2s proliferate to replace lost cells, after which proliferation halts and some AEC2s transdifferentiate into AEC1s to restore normal alveolar structure and function. Although the mechanisms underlying AEC2 proliferation have been studied, the mechanisms responsible for halting proliferation and inducing transdifferentiation are poorly understood. To identify candidate signaling pathways responsible for halting proliferation and inducing transdifferentiation, we performed single cell RNA sequencing on AEC2s during regeneration in a murine model of lung injury induced by intratracheal LPS. Unsupervised clustering revealed distinct subpopulations of regenerating AEC2s: proliferating, cell cycle arrest, and transdifferentiating. Gene expression analysis of these transitional subpopulations revealed that TGFß signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGFß was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGFß is a critical signal halting AEC2 proliferation but must be inactivated to allow transdifferentiation. This study provides insight into the molecular mechanisms regulating alveolar regeneration and the pathogenesis of diseases resulting from a failure of regeneration.


Asunto(s)
Células Epiteliales Alveolares/patología , Lesión Pulmonar/patología , Regeneración , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Lipopolisacáridos/inmunología , Lesión Pulmonar/inmunología , Masculino , Ratones , Cultivo Primario de Células , RNA-Seq , Ratas , Transducción de Señal/fisiología , Análisis de la Célula Individual
14.
Clin Cancer Res ; 25(17): 5242-5249, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31175092

RESUMEN

PURPOSE: With recent advancements in personalized medicine, biopsies must contain sufficient tumor for histologic diagnosis and molecular testing. However, inadvertent biopsy of tumor-associated fibrosis compromises tumor yield, resulting in delayed diagnoses and/or repeat procedures when additional tumor is needed. The ability to differentiate tumor from fibrosis intraprocedurally during biopsy could significantly increase tumor yield. Polarization-sensitive optical coherence tomography (PS-OCT) is an imaging modality that is endoscope- and/or needle-compatible, and provides large volumetric views of tissue microstructure with high resolution (∼10 µm) while simultaneously measuring birefringence of organized tissues such as collagen. We aim to determine whether PS-OCT can accurately detect and distinguish tumor-associated fibrosis from tumor. EXPERIMENTAL DESIGN: PS-OCT was obtained ex vivo in 64 lung nodule samples. PS-OCT birefringence was measured and correlated to collagen content in precisely matched histology, quantified on picrosirius red (PSR) staining. RESULTS: There was a strong positive correlation between PS-OCT measurement of birefringent fibrosis and total collagen content by PSR (r = 0.793; P < 0.001). In addition, PS-OCT was able to accurately classify tumor regions with >20% fibrosis from those with low fibrosis (≤20%) that would likely yield higher tumor content (P < 0.0001). CONCLUSIONS: PS-OCT enables accurate fibrosis detection and can distinguish tumor regions with low fibrosis. PS-OCT has significant potential for clinical impact, as the ability to differentiate tumor from fibrosis could be used to guide intraprocedural tissue sampling in vivo, or for rapid biopsy adequacy assessment ex vivo, to increase diagnostic tumor yield essential for patient care and research.


Asunto(s)
Neoplasias Pulmonares/diagnóstico , Pulmón/patología , Anciano , Anciano de 80 o más Años , Biopsia/métodos , Diagnóstico Diferencial , Femenino , Fibrosis/diagnóstico , Fibrosis/patología , Fibrosis/cirugía , Humanos , Pulmón/cirugía , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Microscopía de Polarización/métodos , Persona de Mediana Edad , Tomografía de Coherencia Óptica/métodos
15.
Stem Cell Reports ; 10(1): 101-119, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29249664

RESUMEN

The current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.


Asunto(s)
Feto , Pulmón , Células Madre Pluripotentes , Animales , Feto/citología , Feto/metabolismo , Xenoinjertos , Humanos , Pulmón/citología , Pulmón/embriología , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante
16.
Nat Cell Biol ; 19(11): 1326-1335, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29058719

RESUMEN

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.


Asunto(s)
Colon/efectos de los fármacos , Hidrogeles/farmacología , Intestinos/efectos de los fármacos , Organoides/efectos de los fármacos , Regeneración/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Humanos , Mucosa Intestinal/efectos de los fármacos , Ratones , Células Madre Pluripotentes/efectos de los fármacos
17.
Nat Microbiol ; 2: 17096, 2017 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-28628099

RESUMEN

Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.


Asunto(s)
Autofagosomas/metabolismo , Interacciones Huésped-Patógeno , Lisosomas/metabolismo , Toxoplasma/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Fibroblastos/parasitología , Técnicas de Inactivación de Genes , Humanos , Ratones Endogámicos C57BL , Neuronas/parasitología , Proteolisis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/metabolismo
18.
Curr Pathobiol Rep ; 4: 47-57, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27340610

RESUMEN

The number and severity of diseases affecting human lung development and adult respiratory function has stimulated great interest in new in vitro models to study the human lung. This review summarizes the most recent breakthroughs deriving lung lineages in a dish by directing the differentiation of human pluripotent stem cells. A variety of culturing platforms have been developed, including two-dimensional and three-dimensional (organoid) culture platforms, to derive specific cell types and structures of the lung. These stem cell-derived lung models will further our understanding of human lung development, disease, and regeneration.

19.
Elife ; 52016 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-27677847

RESUMEN

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung.


Asunto(s)
Diferenciación Celular , Pulmón/citología , Organoides/citología , Células Madre Pluripotentes/fisiología , Poliglactina 910/metabolismo , Andamios del Tejido , Animales , Humanos , Ratones , Trasplantes/citología
20.
Sci Transl Med ; 8(359): 359ra131, 2016 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-27708064

RESUMEN

The inability to visualize airway smooth muscle (ASM) cells in vivo is a major obstacle in understanding their role in normal physiology and diseases. At present, there is no imaging modality available to assess ASM in vivo. Confocal endomicroscopy lacks the penetration depth and field of view, and conventional optical coherence tomography (OCT) does not have sufficient contrast to differentiate ASM from surrounding tissues. We have developed a birefringence microscopy platform that leverages the micro-organization of tissue to add further dimension to traditional OCT. We have used this technology to validate ASM measurements in ex vivo swine and canine studies, visualize and characterize volumetric representations of ASM in vivo, and quantify and predict ASM contractile force as a function of optical retardation. We provide in vivo images and volumetric assessments of ASM in living humans and document structural disease variations in subjects with mild asthma. The opportunity to link inflammatory responses to ASM responses and to link ASM responses to clinical responses and outcomes could lead to an increased understanding of diseases of the airway and, ultimately, to improved patient outcomes.


Asunto(s)
Microscopía/métodos , Músculo Liso/anatomía & histología , Músculo Liso/fisiología , Sistema Respiratorio/anatomía & histología , Animales , Asma/fisiopatología , Birrefringencia , Cartílago/anatomía & histología , Estudios de Casos y Controles , Perros , Humanos , Imagenología Tridimensional , Contracción Muscular , Relajación Muscular , Sus scrofa , Tomografía de Coherencia Óptica
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