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1.
Nature ; 628(8007): 391-399, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38408487

RESUMEN

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.


Asunto(s)
Tipificación del Cuerpo , Microfluídica , Tubo Neural , Humanos , Técnicas de Cultivo Tridimensional de Células , Diferenciación Celular , Cresta Neural/citología , Cresta Neural/embriología , Tubo Neural/citología , Tubo Neural/embriología , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Prosencéfalo/embriología , Médula Espinal/citología , Médula Espinal/embriología
2.
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
3.
JCI Insight ; 8(6)2023 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-36821371

RESUMEN

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.


Asunto(s)
Factor de Crecimiento Epidérmico , Intestinos , Humanos , Epirregulina , Mucosa Intestinal , Diferenciación Celular
4.
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
5.
Development ; 149(20)2022 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-36039869

RESUMEN

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Factor Nuclear Tiroideo 1/metabolismo , Células Epiteliales Alveolares , Diferenciación Celular , Humanos , Pulmón , Organoides
6.
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
7.
Stem Cell Reports ; 17(5): 1138-1153, 2022 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-35395175

RESUMEN

NOTCH signaling is a key regulator involved in maintaining intestinal stem cell (ISC) homeostasis and for balancing differentiation. Using single-cell transcriptomics, we observed that OLFM4, a NOTCH target gene present in ISCs, is first expressed at 13 weeks post-conception in the developing human intestine and increases over time. This led us to hypothesize that the requirement for NOTCH signaling is acquired across human development. To test this, we established a series of epithelium-only organoids (enteroids) from different developmental stages and used γ-secretase inhibitors (dibenzazepine [DBZ] or DAPT) to functionally block NOTCH signaling. Using quantitative enteroid-forming assays, we observed a decrease in enteroid forming efficiency in response to γ-secretase inhibition as development progress. When DBZ was added to cultures and maintained during routine passaging, enteroids isolated from tissue before 20 weeks had higher recovery rates following single-cell serial passaging. Finally, bulk RNA sequencing (RNA-seq) analysis 1 day and 3 days after DBZ treatment showed major differences in the transcriptional changes between developing or adult enteroids. Collectively, these data suggest that ISC dependence on NOTCH signaling increases as the human intestine matures.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Receptores Notch , Células Madre , Secretasas de la Proteína Precursora del Amiloide/genética , Diferenciación Celular , Humanos , Mucosa Intestinal , Intestinos , Organoides , Receptores Notch/genética
8.
Cell Rep ; 38(7): 110379, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35172130

RESUMEN

Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.


Asunto(s)
Epitelio/crecimiento & desarrollo , Intestinos/crecimiento & desarrollo , Organoides/crecimiento & desarrollo , Membrana Serosa/crecimiento & desarrollo , Técnicas de Cultivo de Tejidos , Alginatos/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colágeno/farmacología , Combinación de Medicamentos , Epitelio/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Intestinos/ultraestructura , Laminina/farmacología , Músculo Liso/citología , Organoides/efectos de los fármacos , Organoides/ultraestructura , Proteoglicanos/farmacología , Membrana Serosa/efectos de los fármacos , Membrana Serosa/ultraestructura , Transducción de Señal/efectos de los fármacos , Suspensiones , Proteínas Wnt/metabolismo
9.
Cell ; 184(12): 3281-3298.e22, 2021 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-34019796

RESUMEN

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.


Asunto(s)
Anatomía Artística , Atlas como Asunto , Desarrollo Embrionario , Endodermo/embriología , Modelos Biológicos , Organoides/embriología , Factor de Transcripción CDX2/metabolismo , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Femenino , Gastrulación , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Intestinos/embriología , Masculino , Mesodermo/embriología , Persona de Mediana Edad , Neurregulina-1/metabolismo , Especificidad de Órganos , Células Madre Pluripotentes/citología
10.
Cell Stem Cell ; 28(3): 568-580.e4, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33278341

RESUMEN

The human intestinal stem cell niche supports self-renewal and epithelial function, but little is known about its development. We used single-cell mRNA sequencing with in situ validation approaches to interrogate human intestinal development from 7-21 weeks post conception, assigning molecular identities and spatial locations to cells and factors that comprise the niche. Smooth muscle cells of the muscularis mucosa, in close proximity to proliferative crypts, are a source of WNT and RSPONDIN ligands, whereas EGF is expressed far from crypts in the villus epithelium. Instead, an PDGFRAHI/F3HI/DLL1HI mesenchymal population lines the crypt-villus axis and is the source of the epidermal growth factor (EGF) family member NEUREGULIN1 (NRG1). In developing intestine enteroid cultures, NRG1, but not EGF, permitted increased cellular diversity via differentiation of secretory lineages. This work highlights the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine.


Asunto(s)
Intestinos , Nicho de Células Madre , Diferenciación Celular , Humanos , Mucosa Intestinal , Células Madre
11.
Development ; 147(20)2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-32994164

RESUMEN

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.


Asunto(s)
Sistema Digestivo/crecimiento & desarrollo , Sistema Digestivo/metabolismo , Seudópodos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Masculino , Mesodermo/metabolismo , Ratones Endogámicos C57BL , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo
13.
Dev Cell ; 54(4): 516-528.e7, 2020 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-32841595

RESUMEN

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) lack some cellular populations found in the native organ, including vasculature. Using single-cell RNA sequencing (scRNA-seq), we have identified a population of endothelial cells (ECs) present early in HIO differentiation that declines over time in culture. Here, we developed a method to expand and maintain this endogenous population of ECs within HIOs (vHIOs). Given that ECs possess organ-specific gene expression, morphology, and function, we used bulk RNA-seq and scRNA-seq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC gene signatures. By comparing these gene signatures and validated markers to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data demonstrate that HIOs can co-differentiate a native EC population that is properly patterned with an intestine-specific EC transcriptional signature in vitro.


Asunto(s)
Células Endoteliales/metabolismo , Mucosa Intestinal/crecimiento & desarrollo , Intestinos/crecimiento & desarrollo , Especificidad de Órganos/genética , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Intestinal/metabolismo , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , RNA-Seq
14.
Methods Cell Biol ; 159: 143-174, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32586441

RESUMEN

Human intestinal organoids (HIOs) derived from pluripotent stem cells were first described almost a decade ago as a method to differentiate intestinal tissue containing both epithelium and supporting mesenchymal cells. The original protocol documents a directed differentiation approach to first induce definitive endoderm from pluripotent stem cells, followed by hindgut specification, resulting in the self-organization of 3D hindgut spheroids. These hindgut spheroids are then embedded in a basement membrane extracellular matrix (ECM) such as Matrigel and mature into HIOs over about 4 weeks in culture. Since the initial HIO protocol was published, the methods to generate HIOs have been updated over time including revisions to the directed differentiation protocol and implementation of new culture methods for spheroids such as embedding in alginate or polyethylene glycol hydrogels as defined alternatives to Matrigel. Additionally, HIOs have been utilized for new applications such as co-culture with bacteria. This protocol compiles the most up to date information on HIO generation and presents alternative experimental applications.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Intestino Delgado/citología , Intestino Delgado/fisiología , Organoides/citología , Alginatos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colágeno/farmacología , Combinación de Medicamentos , Endodermo/citología , Humanos , Hidrogeles/farmacología , Laminina/farmacología , Organoides/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Proteoglicanos/farmacología , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos
15.
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
16.
Development ; 146(19)2019 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-31345929

RESUMEN

As embryos mature, cells undergo remarkable transitions that are accompanied by shifts in transcription factor regulatory networks. Mechanisms driving developmental transitions are incompletely understood. The embryonic intestine transitions from a rapidly proliferating tube with pseudostratified epithelium prior to murine embryonic day (E) 14.5 to an exquisitely folded columnar epithelium in fetal stages. We sought to identify factors driving mouse fetal intestinal maturation by mining chromatin accessibility data for transcription factor motifs. ATAC-seq accessible regions shift during tissue maturation, with CDX2 transcription factor motifs abundant at chromatin-accessible regions of the embryo. Hepatocyte nuclear factor 4 (HNF4) transcription factor motifs are the most abundant in the fetal stages (>E16.5). Genetic inactivation of Hnf4a and its paralog Hnf4g revealed that HNF4 factors are redundantly required for fetal maturation. CDX2 binds to and activates Hnf4 gene loci to elevate HNF4 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic regulatory sites to promote chromatin accessibility and gene expression in the maturing intestine. Thus, HNF4 paralogs are key components of an intestinal transcription factor network shift during the embryonic to fetal transition.


Asunto(s)
Cromatina/metabolismo , Feto/embriología , Factor Nuclear 4 del Hepatocito/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/metabolismo , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Morfogénesis
17.
Development ; 146(5)2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30745430

RESUMEN

Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic loss of CDX2 triggers homeotic transformation of intestinal fate, whereas adult-onset loss compromises crucial physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program; rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or mature intestinal function. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.


Asunto(s)
Factor de Transcripción CDX2/metabolismo , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/genética , Sistemas CRISPR-Cas , Diferenciación Celular , Linaje de la Célula , Femenino , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Mutación , Células Madre Pluripotentes/citología , Unión Proteica , Dominios Proteicos , Transactivadores/metabolismo
18.
Stem Cell Reports ; 12(2): 381-394, 2019 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-30612954

RESUMEN

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell instructive properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. In addition, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.


Asunto(s)
Alginatos/farmacología , Hidrogeles/farmacología , Intestinos/efectos de los fármacos , Organoides/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colágeno/farmacología , Combinación de Medicamentos , Epitelio/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Humanos , Laminina/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteoglicanos/farmacología , Ingeniería de Tejidos/métodos
20.
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
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