Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

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.

Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.

Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration.

The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

In vivo directed differentiation of pluripotent stem cells for skeletal regeneration.

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing.

Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (ß-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

The seed and the soil: optimizing stem cells and their environment for tissue regeneration.

The potential for stem cells to serve as cellular building blocks for reconstruction of complex defects has prompted significant enthusiasm in the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly hinder their tissue forming capacity. To circumvent this obstacle, novel approaches have been developed to manipulate both the stem cell itself and its surrounding environmental niche. By understanding this paradigm of seed and soil optimization, innovative strategies may thus be developed to harness the true promise of stem cells for tissue regeneration.

Single-cell transcriptomics of a dynamic cell behavior in murine airways.

Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.

CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor ß1 (TGF-ß1) signaling.

Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-ß1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects.

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.

Nonintegrating knockdown and customized scaffold design enhances human adipose-derived stem cells in skeletal repair.

An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.

Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone.

BACKGROUND: Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration. METHODS: Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur. RESULTS: Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699 ± 27 µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182 ± 22 µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects. CONCLUSIONS: We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

Models of cranial suture biology.

Craniosynostosis is a common congenital defect caused by premature fusion of cranial sutures. The severe morphologic abnormalities and cognitive deficits resulting from craniosynostosis and the potential morbidity of surgical correction espouse the need for a deeper understanding of the complex etiology for this condition. Work in animal models for the past 20 years has been pivotal in advancing our understanding of normal suture biology and elucidating pathologic disease mechanisms. This article provides an overview of milestone studies in suture development, embryonic origins, and signaling mechanisms from an array of animal models including transgenic mice, rats, rabbits, fetal sheep, zebrafish, and frogs. This work contributes to an ongoing effort toward continued development of novel treatment strategies.

Pierre Robin sequence and Treacher Collins hypoplastic mandible comparison using three-dimensional morphometric analysis.

Pierre Robin sequence and Treacher Collins syndrome are both associated with mandibular hypoplasia. It has been hypothesized, however, that the mandible may be differentially affected. The purpose of this study was to therefore compare mandibular morphology in children with Pierre Robin sequence with children with Treacher Collins syndrome using three-dimensional analysis of computed tomographic scans. A retrospective analysis was performed identifying children with Pierre Robin sequence and Treacher Collins syndrome undergoing computed tomography. Three-dimensional reconstruction was performed, and ramus height, mandibular body length, and gonial angle were measured. These were then compared with those in control children with normal mandibles and with the clinical norms corrected for age and sex based on previously published measurements. Mandibular body length was found to be significantly shorter for children with Pierre Robin sequence, whereas ramus height was significantly shorter for children with Treacher Collins syndrome. This resulted in distinctly different ramus height-mandibular body length ratios. In addition, the gonial angle was more obtuse in both the Pierre Robin sequence and Treacher Collins syndrome groups compared with the controls. Three-dimensional mandibular morphometric analysis in patients with Pierre Robin sequence and Treacher Collins syndrome thus revealed distinctly different patterns of mandibular hypoplasia relative to normal controls. These findings underscore distinct considerations that must be made in surgical planning for reconstruction.

Identifying disease-critical cell types and cellular processes by integrating single-cell RNA-sequencing and human genetics.

Genome-wide association studies provide a powerful means of identifying loci and genes contributing to disease, but in many cases, the related cell types/states through which genes confer disease risk remain unknown. Deciphering such relationships is important for identifying pathogenic processes and developing therapeutics. In the present study, we introduce sc-linker, a framework for integrating single-cell RNA-sequencing, epigenomic SNP-to-gene maps and genome-wide association study summary statistics to infer the underlying cell types and processes by which genetic variants influence disease. The inferred disease enrichments recapitulated known biology and highlighted notable cell-disease relationships, including γ-aminobutyric acid-ergic neurons in major depressive disorder, a disease-dependent M-cell program in ulcerative colitis and a disease-specific complement cascade process in multiple sclerosis. In autoimmune disease, both healthy and disease-dependent immune cell-type programs were associated, whereas only disease-dependent epithelial cell programs were prominent, suggesting a role in disease response rather than initiation. Our framework provides a powerful approach for identifying the cell types and cellular processes by which genetic variants influence disease.

Identifying disease-critical cell types and cellular processes across the human body by integration of single-cell profiles and human genetics.

Genome-wide association studies (GWAS) provide a powerful means to identify loci and genes contributing to disease, but in many cases the related cell types/states through which genes confer disease risk remain unknown. Deciphering such relationships is important for identifying pathogenic processes and developing therapeutics. Here, we introduce sc-linker, a framework for integrating single-cell RNA-seq (scRNA-seq), epigenomic maps and GWAS summary statistics to infer the underlying cell types and processes by which genetic variants influence disease. We analyzed 1.6 million scRNA-seq profiles from 209 individuals spanning 11 tissue types and 6 disease conditions, and constructed gene programs capturing cell types, disease progression, and cellular processes both within and across cell types. We evaluated these gene programs for disease enrichment by transforming them to SNP annotations with tissue-specific epigenomic maps and computing enrichment scores across 60 diseases and complex traits (average N= 297K). Cell type, disease progression, and cellular process programs captured distinct heritability signals even within the same cell type, as we show in multiple complex diseases that affect the brain (Alzheimer’s disease, multiple sclerosis), colon (ulcerative colitis) and lung (asthma, idiopathic pulmonary fibrosis, severe COVID-19). The inferred disease enrichments recapitulated known biology and highlighted novel cell-disease relationships, including GABAergic neurons in major depressive disorder (MDD), a disease progression M cell program in ulcerative colitis, and a disease-specific complement cascade process in multiple sclerosis. In autoimmune disease, both healthy and disease progression immune cell type programs were associated, whereas for epithelial cells, disease progression programs were most prominent, perhaps suggesting a role in disease progression over initiation. Our framework provides a powerful approach for identifying the cell types and cellular processes by which genetic variants influence disease.