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/citologíaRESUMEN
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/metabolismoRESUMEN
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 CelularRESUMEN
Frequent injections of anti-CD124 monoclonal antibody (αCD124) over long periods of time are used to treat chronic rhinosinusitis with nasal polyps (CRSwNP). Needle-free, intranasal administration (i.n.) of αCD124 is expected to provide advantages of localized delivery, improved efficacy, and enhanced medication adherence. However, delivery barriers such as the mucus and epithelium in the nasal tissue impede penetration of αCD124. Herein, two novel protamine nanoconstructs: allyl glycidyl ether conjugated protamine (Nano-P) and polyamidoamine-linked protamine (Dendri-P) were synthesized and showed enhanced αCD124 penetration through multiple epithelial layers compared to protamine in mice. αCD124 was mixed with Nano-P or Dendri-P and then intranasally delivered for the treatment of severe CRSwNP in mice. Micro-CT and pathological changes in nasal turbinates showed that these two nano-formulations achieved â¼50 % and â¼40 % reductions in nasal polypoid lesions and eosinophil count, respectively. Both nano-formulations provided enhanced efficacy in suppressing nasal and systemic Immunoglobulin E (IgE) and nasal type 2 inflammatory biomarkers, such as interleukin 13 (IL-13) and IL-25. These effects were superior to those in the protamine formulation group and subcutaneous (s.c.) αCD124 given at a 12.5-fold higher dose. Intranasal delivery of protamine, Nano-P, or Dendri-P did not induce any measurable toxicities in mice.
Asunto(s)
Anticuerpos Monoclonales , Pólipos Nasales , Protaminas , Rinosinusitis , Animales , Femenino , Ratones , Administración Intranasal , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Enfermedad Crónica , Ratones Endogámicos BALB C , Pólipos Nasales/tratamiento farmacológico , Pólipos Nasales/patología , Protaminas/química , Rinosinusitis/tratamiento farmacológicoRESUMEN
Valve remodeling is a process involving extracellular matrix organization and elongation of valve leaflets. Here, through single-cell RNA sequencing of human fetal valves, we identified an elastin-producing valve interstitial cell (VIC) subtype (apolipoprotein E (APOE)+, elastin-VICs) spatially located underneath valve endothelial cells (VECs) sensing unidirectional flow. APOE knockdown in fetal VICs resulted in profound elastogenesis defects. In valves with pulmonary stenosis (PS), we observed elastin fragmentation and decreased expression of APOE along with other genes regulating elastogenesis. Cell-cell interaction analysis revealed that jagged 1 (JAG1) from unidirectional VECs activates elastogenesis in elastin-VICs through NOTCH2. Similar observations were made in VICs cocultured with VECs under unidirectional flow. Notably, a drastic reduction of JAG1-NOTCH2 was also observed in PS valves. Lastly, we found that APOE controls JAG1-induced NOTCH activation and elastogenesis in VICs through the extracellular signal-regulated kinase pathway. Our study suggests important roles of both APOE and NOTCH in regulating elastogenesis during human valve remodeling.
Asunto(s)
Apolipoproteínas E , Elastina , Células Endoteliales , Proteína Jagged-1 , Transducción de Señal , Humanos , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Elastina/metabolismo , Elastina/genética , Células Endoteliales/metabolismo , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Receptor Notch2/metabolismo , Receptor Notch2/genética , Células Cultivadas , Válvula Pulmonar/metabolismo , Técnicas de Cocultivo , Comunicación Celular/fisiología , Válvulas Cardíacas/embriología , Válvulas Cardíacas/metabolismoRESUMEN
Proteins and peptides often require frequent needle-based administrations. Here, we report a non-parenteral delivery method for proteins through physical mixing with protamine, an FDA-approved peptide. Protamine was shown to promote tubulation and rearrangement of cellular actin, leading to enhanced intracellular delivery of proteins compared to poly(arginine)8 (R8). While the R8-mediated delivery resulted in significant lysosomal accumulation of the cargo, protamine directed the proteins to the nuclei with little lysosomal uptake. Intranasal delivery of insulin mixed with protamine effectively reduced blood glucose levels in diabetic mice 0.5 h after administration and the effect lasted for â¼6 h, comparable to subcutaneously injected insulin at the same dose. In mice, protamine was shown to overcome mucosal and epithelial barriers and modulate adherens junctions, promoting insulin penetration to the lamina propria layer for systemic absorption.
Asunto(s)
Péptidos de Penetración Celular , Diabetes Mellitus Experimental , Ratones , Animales , Protaminas , Diabetes Mellitus Experimental/tratamiento farmacológico , InsulinaRESUMEN
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 CelularRESUMEN
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/genéticaRESUMEN
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/metabolismoRESUMEN
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 WntRESUMEN
BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.
Asunto(s)
Adenocarcinoma , Esófago de Barrett , Neoplasias Esofágicas , Proteína p53 Supresora de Tumor , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Adenocarcinoma/patología , Esófago de Barrett/genética , Esófago de Barrett/patología , Progresión de la Enfermedad , Neoplasias Esofágicas/patología , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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 MadreRESUMEN
Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.
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/métodosRESUMEN
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-SeqRESUMEN
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.