A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates.

We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling.

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

RTK signalling promotes epithelial columnar cell shape and apical junction maintenance in human lung progenitor cells.

Multipotent epithelial progenitor cells can be expanded from human embryonic lungs as organoids and maintained in a self-renewing state using a defined medium. The organoid cells are columnar, resembling the cell morphology of the developing lung tip epithelium in vivo. Cell shape dynamics and fate are tightly coordinated during development. We therefore used the organoid system to identify signalling pathways that maintain the columnar shape of human lung tip progenitors. We found that EGF, FGF7 and FGF10 have distinct functions in lung tip progenitors. FGF7 activates MAPK/ERK and PI3K/AKT signalling, and is sufficient to promote columnar cell shape in primary tip progenitors. Inhibitor experiments show that MAPK/ERK and PI3K/AKT signalling are key downstream pathways, regulating cell proliferation, columnar cell shape and cell junctions. We identified integrin signalling as a key pathway downstream of MAPK/ERK in the tip progenitors; disrupting integrin alters polarity, cell adhesion and tight junction assembly. By contrast, stimulation with FGF10 or EGF alone is not sufficient to maintain organoid columnar cell shape. This study employs organoids to provide insight into the cellular mechanisms regulating human lung development.

Exploring the challenges and opportunities of public engagement with fundamental biology.

The Human Developmental Biology Initiative (HDBI) is a Wellcome-funded research consortium involving scientists based in institutions across the UK and Europe. It aims to pioneer new technologies and techniques to answer fundamental questions about human development and could, therefore, eventually improve treatments for fertility, birth defects and developmental diseases, as well as aiding regenerative medicine. HDBI research relies on human fetal and embryonic tissues donated following pregnancy terminations or fertility treatment. The situations in which these tissues are donated, their use in research and the potential healthcare impacts of this work all present complex ethical and moral questions that are of interest not only to scientists but also to the public. As such, HDBI's public engagement programme 'What makes us human?' aspires to test new ways of engaging the public with fundamental biology. In this brief Perspective, we provide an overview of this public engagement approach, exploring its challenges and opportunities, and outline our longer-term plans. We hope that by sharing our experiences we will encourage and enable others to organise similarly experimental public engagement, even if their research is very fundamental or potentially controversial.

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Human lung development: recent progress and new challenges.

Recent studies have revealed biologically significant differences between human and mouse lung development, and have reported new in vitro systems that allow experimental manipulation of human lung models. At the same time, emerging clinical data suggest that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. In this Review, we discuss our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments using in vitro human lung developmental models and RNA sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on the mechanisms underlying human lung development and disease, and may help pave the way for new therapies.

The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells.

Innate lymphoid cells (ILCs) reside at mucosal surfaces and control immunity to intestinal infections. Type 2 innate lymphoid cells (ILC2s) produce cytokines such as IL-5 and IL-13, are required for immune defense against helminth infections, and are involved in the pathogenesis of airway hyperreactivity. Here, we have investigated the role of the transcription factor GATA-3 for ILC2 differentiation and maintenance. We showed that ILC2s and their lineage-specified bone marrow precursors (ILC2Ps), as identified here, were characterized by continuous high expression of GATA-3. Analysis of mice with temporary deletion of GATA-3 in all ILCs showed that GATA-3 was required for the differentiation and maintenance of ILC2s but not for RORγt(+) ILCs. Thus, our data demonstrate that GATA-3 is essential for ILC2 fate decisions and reveal similarities between the transcriptional programs controlling ILC and T helper cell fates.

FGFR2 is required for airway basal cell self-renewal and terminal differentiation.

Airway stem cells slowly self-renew and produce differentiated progeny to maintain homeostasis throughout the lifespan of an individual. Mutations in the molecular regulators of these processes may drive cancer or degenerative disease, but are also potential therapeutic targets. Conditionally deleting one copy of FGF receptor 2 (FGFR2) in adult mouse airway basal cells results in self-renewal and differentiation phenotypes. We show that FGFR2 signalling correlates with maintenance of expression of a key transcription factor for basal cell self-renewal and differentiation: SOX2. This heterozygous phenotype illustrates that subtle changes in receptor tyrosine kinase signalling can have significant effects, perhaps providing an explanation for the numerous changes seen in cancer.

A Subpopulation of Foxj1-Expressing, Nonmyelinating Schwann Cells of the Peripheral Nervous System Contribute to Schwann Cell Remyelination in the Central Nervous System.

New myelin sheaths can be restored to demyelinated axons in a spontaneous regenerative process called remyelination. In general, new myelin sheaths are made by oligodendrocytes newly generated from a widespread population of adult CNS progenitors called oligodendrocyte progenitor cells (OPCs). New myelin in CNS remyelination in both experimental models and clinical diseases can also be generated by Schwann cells (SCs), the myelin-forming cells of the PNS. Fate-mapping studies have shown that SCs contributing to remyelination in the CNS are often derived from OPCs and appear not to be derived from myelinating SCs from the PNS. In this study, we address whether CNS remyelinating SCs can also be generated from PNS-derived cells other than myelinating SCs. Using a genetic fate-mapping approach, we have found that a subpopulation of nonmyelinating SCs identified by the expression of the transcription factor Foxj1 also contribute to CNS SC remyelination, as well as to remyelination in the PNS. We also find that the ependymal cells lining the central canal of the spinal cord, which also express Foxj1, do not generate cells that contribute to CNS remyelination. These findings therefore identify a previously unrecognized population of PNS glia that can participate in the regeneration of new myelin sheaths following CNS demyelination.SIGNIFICANCE STATEMENT Remyelination failure in chronic demyelinating diseases such as multiple sclerosis drives the current quest for developing means by which remyelination in CNS can be enhanced therapeutically. Critical to this endeavor is the need to understand the mechanisms of remyelination, including the nature and identity of the cells capable of generating new myelin sheath-forming cells. Here, we report a previously unrecognized subpopulation of nonmyelinating Schwann cells (SCs) in the PNS, identified by the expression of the transcription factor Foxj1, which can give rise to SCs that are capable of remyelinating both PNS and CNS axons. These cells therefore represent a new cellular target for myelin regenerative strategies for the treatment of CNS disorders characterized by persistent demyelination.

The Human Lung Cell Atlas: A High-Resolution Reference Map of the Human Lung in Health and Disease.

Lung disease accounts for every sixth death globally. Profiling the molecular state of all lung cell types in health and disease is currently revolutionizing the identification of disease mechanisms and will aid the design of novel diagnostic and personalized therapeutic regimens. Recent progress in high-throughput techniques for single-cell genomic and transcriptomic analyses has opened up new possibilities to study individual cells within a tissue, classify these into cell types, and characterize variations in their molecular profiles as a function of genetics, environment, cell-cell interactions, developmental processes, aging, or disease. Integration of these cell state definitions with spatial information allows the in-depth molecular description of cellular neighborhoods and tissue microenvironments, including the tissue resident structural and immune cells, the tissue matrix, and the microbiome. The Human Cell Atlas consortium aims to characterize all cells in the healthy human body and has prioritized lung tissue as one of the flagship projects. Here, we present the rationale, the approach, and the expected impact of a Human Lung Cell Atlas.

Developmental mechanisms and adult stem cells for therapeutic lung regeneration.

Chronic degenerative lung diseases are essentially untreatable pathological conditions. By contrast, the healthy lung has numerous mechanisms that allow for rapid repair and restoration of function following minor acute injuries. We discuss the normal endogenous processes of lung development, homeostatic maintenance and repair and consider the research strategies required for the development of methods for human therapeutic lung regeneration.

Inhibition of ß-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing.

New hair follicles (HFs) do not form in adult mammalian skin unless epidermal Wnt signalling is activated genetically or within large wounds. To understand the postnatal loss of hair forming ability we monitored HF formation at small circular (2 mm) wound sites. At P2, new HFs formed in back skin, but HF formation was markedly decreased by P21. Neonatal tail also formed wound-associated HFs, albeit in smaller numbers. Postnatal loss of HF neogenesis did not correlate with wound closure rate but with a reduction in Lrig1-positive papillary fibroblasts in wounds. Comparative gene expression profiling of back and tail dermis at P1 and dorsal fibroblasts at P2 and P50 showed a correlation between loss of HF formation and decreased expression of genes associated with proliferation and Wnt/ß-catenin activity. Between P2 and P50, fibroblast density declined throughout the dermis and clones of fibroblasts became more dispersed. This correlated with a decline in fibroblasts expressing a TOPGFP reporter of Wnt activation. Surprisingly, between P2 and P50 there was no difference in fibroblast proliferation at the wound site but Wnt signalling was highly upregulated in healing dermis of P21 compared with P2 mice. Postnatal ß-catenin ablation in fibroblasts promoted HF regeneration in neonatal and adult mouse wounds, whereas ß-catenin activation reduced HF regeneration in neonatal wounds. Our data support a model whereby postnatal loss of hair forming ability in wounds reflects elevated dermal Wnt/ß-catenin activation in the wound bed, increasing the abundance of fibroblasts that are unable to induce HF formation.

Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate.

Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity.

SOX2 Drives Bronchial Dysplasia in a Novel Organotypic Model of Early Human Squamous Lung Cancer.

RATIONALE: Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of sex-determining region Y-box 2 (SOX2) is an early and consistent event in the pathogenesis of this disease, but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs. OBJECTIVES: (1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease; (2) to test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia; and (3) to use the model for studies on pathogenesis and chemoprevention. METHODS: We engineered the inducible activation of oncogenes in immortalized bronchial epithelial cells. We used three-dimensional tissue culture to build an organotypic model of bronchial dysplasia. MEASUREMENTS AND MAIN RESULTS: We recapitulated human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of tumor promoter 53 is a cooperating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype. CONCLUSIONS: In the appropriate genetic and microenvironmental context, acute deregulation of SOX2 drives bronchial dysplasia. This confirms its oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.

Intervention fidelity in a school-based diet and physical activity intervention in the UK: Active for Life Year 5.

BACKGROUND: Active for Life Year 5 (AFLY5) is an educational programme for Year 5 children (aged 9-10) designed to increase children's physical activity, decrease sedentary behaviour and increase fruit and vegetable intake. This paper reports findings from a process evaluation embedded within a randomised controlled trial evaluating the programme's effectiveness. It considers the fidelity of implementation of AFLY5 with a focus on three research questions: 1. To what extent was the intervention delivered as planned? 2. In what ways, if any, did the teachers amend the programme? and 3. What were the reasons for any amendments? METHODS: Mixed methods were used including data collection via observation of the intervention delivery, questionnaire, teacher's intervention delivery log and semi-structured interviews with teachers and parents. Qualitative data were analysed thematically and quantitative data were summarised using descriptive statistics. RESULTS: Following training, 42 of the 43 intervention school teachers/teaching staff (98%) were confident they could deliver the nutrition and physical activity lessons according to plan. The mean number of lessons taught was 12.3 (s.d. 3.7), equating to 77% of the intervention. Reach was high with 95% of children in intervention schools receiving lessons. A mean of 6.2 (s.d. 2.6) out of 10 homeworks were delivered. Median lesson preparation time was 10 min (IQR 10-20) and 28% of lessons were reported as having been amended. Qualitative findings revealed that those who amended the lessons did so to differentiate for student ability, update them for use with new technologies and to enhance teacher and student engagement. Teachers endorsed the aims of the intervention, but some were frustrated with having to adapt the lesson materials. Teachers also a reported tendency to delegate the physical activity lessons to other staff not trained in the intervention. CONCLUSIONS: Fidelity of intervention implementation was good but teachers' enthusiasm for the AFLY5 programme was mixed despite them believing that the messages behind the lessons were important. This may have meant that the intervention messages were not delivered as anticipated and explain why the intervention was found not to be effective. TRIAL REGISTRATION: ISRCTN50133740.

Deterministic nuclear reprogramming of mammalian nuclei to a totipotency-like state by Amphibian meiotic oocytes for stem cell therapy in humans.

The ultimate aim of nuclear reprogramming is to provide stem cells or differentiated cells from unrelated cell types as a cell source for regenerative medicine. A popular route towards this is transcription factor induction, and an alternative way is an original procedure of transplanting a single somatic cell nucleus to an unfertilized egg. A third route is to transplant hundreds of cell nuclei into the germinal vesicle (GV) of a non-dividing Amphibian meiotic oocyte, which leads to the activation of silent genes in 24 h and robustly induces a totipotency-like state in almost all transplanted cells. We apply this third route for potential therapeutic use and describe a procedure by which the differentiated states of cells can be reversed so that totipotency and pluripotency gene expression are regained. Differentiated cells are exposed to GV extracts and are reprogrammed to form embryoid bodies, which shows the maintenance of stemness and could be induced to follow new directions of differentiation. We conclude that much of the reprogramming effect of eggs is already present in meiotic oocytes and does not require cell division or selection of dividing cells. Reprogrammed cells by oocytes could serve as replacements for defective adult cells in humans.

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease.

Alveolar type 2 (AT2) cells maintain lung health by acting as stem cells and producing pulmonary surfactant1-3. AT2 dysfunction underlies many lung diseases including interstitial lung disease (ILD), in which some inherited forms result from mislocalisation of surfactant protein C (SFTPC) variants4,5. Disease modelling and dissection of mechanisms remains challenging due to complexities in deriving and maintaining AT2 cells ex vivo. Here, we describe the development of expandable adult AT2-like organoids derived from human fetal lung which are phenotypically stable, can differentiate into AT1-like cells and are genetically manipulable. We use these organoids to test key effectors of SFTPC maturation identified in a forward genetic screen including the E3 ligase ITCH, demonstrating that their depletion phenocopies the pathological SFTPC redistribution seen for the SFTPC-I73T variant. In summary, we demonstrate the development of a novel alveolar organoid model and use it to identify effectors of SFTPC maturation necessary for AT2 health.