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Project description:Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
| S-EPMC5995784 | biostudies-other
Project description:Many lung diseases involve alterations in the cellular identity of the lung epithelium. Improved insight into airway development and the changes to cellular identity that result from abnormal signaling may therefore improve understanding of the etiology of these complex diseases. We have previously described a protocol to generate epithelial airway spheres from human pluripotent stem cells (hPSCs), but still poorly understand the identity, development, and heterogeneity of these early airway cells. Here we use novel murine and human PSC lines to study developing secretory cells derived in vitro from hPSCs. Using an SCGB3A2CherryPicker (SC) reporter system together with population-based or single-cell global transcriptomic profiling we track, purify, and analyze hPSC-derived putative secretory airway progenitors and find that SC+ cells are enriched for expression of airway epithelial markers, including secretory cell markers, SCGB3A2 and SCGB1A1. Unexpectedly, some SC+ human cells also co-express distal type 2 cell genes, including SFTPC, ABCA3, NAPSA, CTSH and functional lamellar bodies, suggesting a significant level of plasticity within the hPSC-derived secretory population relative to concurrently generated proximal airway TP63+ cells or distal alveolar SFTPC+ cells. We establish that this plasticity is minimized by inhibiting low levels of endogenous canonical Wnt signaling post-lung specification, thus depleting the co-expressed type 2 cell program. Taken together, these findings suggest that, similar to in vivo mouse genetic models and diseased human lungs, hPSC-derived airway cells exhibit cellular plasticity in response to signaling cues, providing a human model system in which to study cellular identity in a disease-relevant context.
2018-02-28 | GSE103919 | GEO
Project description:Many lung diseases involve alterations in the cellular identity of the lung epithelium. Improved insight into airway development and the changes to cellular identity that result from abnormal signaling may therefore improve understanding of the etiology of these complex diseases. We have previously described a protocol to generate epithelial airway spheres from human pluripotent stem cells (hPSCs), but still poorly understand the identity, development, and heterogeneity of these early airway cells. Here we use novel murine and human PSC lines to study developing secretory cells derived in vitro from hPSCs. Using an SCGB3A2CherryPicker (SC) reporter system together with population-based or single-cell global transcriptomic profiling we track, purify, and analyze hPSC-derived putative secretory airway progenitors and find that SC+ cells are enriched for expression of airway epithelial markers, including secretory cell markers, SCGB3A2 and SCGB1A1. Unexpectedly, some SC+ human cells also co-express distal type 2 cell genes, including SFTPC, ABCA3, NAPSA, CTSH and functional lamellar bodies, suggesting a significant level of plasticity within the hPSC-derived secretory population relative to concurrently generated proximal airway TP63+ cells or distal alveolar SFTPC+ cells. We establish that this plasticity is minimized by inhibiting low levels of endogenous canonical Wnt signaling post-lung specification, thus depleting the co-expressed type 2 cell program. Taken together, these findings suggest that, similar to in vivo mouse genetic models and diseased human lungs, hPSC-derived airway cells exhibit cellular plasticity in response to signaling cues, providing a human model system in which to study cellular identity in a disease-relevant context.
2018-02-28 | GSE103918 | GEO
Project description:Single cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium reveals fate plasticity
| PRJNA407478 | ENA
Project description:We sought to better understand the kinetics of differentiation and the global transcriptomic programs of these labeled cells as they emerge over prolonged culture periods
2018-04-12 | GSE103517 | GEO
Project description:Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of diseases associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate time points of retinal pigment epithelium (RPE) cultures that reflect native counterparts, we characterised the transcriptomic profiles of the hPSC-derived RPE cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single-cell RNA-sequencing of a total of 16,576 cells to assess the molecular changes of the RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial-mesenchymal transition, and with the maturing populations of the RPE observed with time in culture. Assessment of Gene Ontology pathways revealed that as the cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirms a maturing transcriptional profile of RPE cells in culture. These results suggest that long-term in vitro culture of RPE cells allows the modelling of specific phenotypes observed in native mature tissues. Our work highlights the transcriptional landscape of hPSC-derived RPE cells as they age in culture, which provides a reference for native and patient samples to be benchmarked against.
| S-EPMC8602392 | biostudies-literature
Project description:Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.
| S-EPMC7923590 | biostudies-literature
Project description:About 50% of patients with asthma exhibit chronic airway inflammation driven by the type 2 cytokines interleukin (IL)-4, IL-5, and IL-13. These patients with type 2-high asthma experience more allergic symptoms, greater airway hyperresponsiveness, and more severe mucus obstruction than patients with type 2-low asthma. Mouse models of asthma have shown that much of the airway dysfunction in these models can be generated by IL-13 stimulation of the airway epithelium alone. Both in vivo mouse model studies and in vitro studies of human mucociliary airway epithelial cultures have shown that IL-13 induces cellular remodeling of the airway epithelium through proliferation-independent transdifferentiation processes. In both humans and mice, IL-13 stimulation of the airway epithelium results in generation of hypersecretory mucin 5AC (MUC5AC)-expressing mucus cells. Whereas club cells have been shown to be the source of these mucin 5AC-positive mucus cells in mice, the origin of these mucus cells in humans is unclear. In humans, chronic IL-13 stimulation appears to result in loss of ciliated cells. Moreover, IL-13 stimulation can block ciliated cell differentiation from human basal airway epithelial cells. Coincident with IL-13 cellular remodeling are reported decreases in mucociliary transport and ciliary beat frequency. These IL-13-mediated changes in mucociliary function are accompanied by disorganization of cilia, a decrease in the ratio of mucin 5B (MUC5B) to mucin 5AC, and mucus gel tethering to the epithelial surface by mucin 5AC. These airway epithelial responses to IL-13 are mediated by multiple transcription factors, including signal transducer and activator of transcription-6 (STAT6), SAM pointed domain-containing Ets transcription factor (SPDEF), Forkhead box A2 (FOXA2), and Forkhead box J1 (FOXJ1). In addition, analysis of RNA-sequencing data derived from airway epithelial cells shows how IL-13 stimulation promotes broad changes in gene expression across the transcriptome. These results reveal the plastic nature of airway epithelial cells that enables the epithelium to undergo remodeling and functional shifts in response to IL-13 stimulation. With use of new technology, future studies should lead to greater understanding of how IL-13 and other stimuli of disease bring about airway epithelial remodeling, which may aid in the development of therapies that ameliorate airway dysfunction in asthma and other diseases.
| S-EPMC5955044 | biostudies-literature
Project description:Background Bronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles. Methods BASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated. Results Flow cytometric analysis revealed that BASCs represented ~?7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs. Conclusions Mouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.
| S-EPMC7531137 | biostudies-literature
Project description:Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-? pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture.
| S-EPMC4149306 | biostudies-literature