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Nature Cell Biology Jan 2022Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis...
Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-β1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1/TGFB1 fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.
Topics: Alveolar Epithelial Cells; Animals; Bone Morphogenetic Proteins; Cell Differentiation; Cell Transdifferentiation; Cells, Cultured; Epidermal Cells; Epithelial Cells; Fibroblasts; Humans; Idiopathic Pulmonary Fibrosis; Keratin-5; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Pulmonary Alveoli; Respiratory Mucosa; Signal Transduction; Single-Cell Analysis; Transforming Growth Factor beta1
PubMed: 34969962
DOI: 10.1038/s41556-021-00809-4 -
Nature Biotechnology Aug 2019Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel...
Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8 T cell exhaustion and CD4 T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.
Topics: Bone Marrow Cells; Cell Line; Chromatin; Computer Simulation; Gene Expression Regulation; Hematopoiesis; High-Throughput Nucleotide Sequencing; Humans; Leukocytes, Mononuclear; Single-Cell Analysis; T-Lymphocytes; Transcription Factors
PubMed: 31375813
DOI: 10.1038/s41587-019-0206-z -
American Journal of Respiratory and... Dec 2020Declining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is...
Declining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF). We sought to decipher the transcriptome of freshly isolated epithelial cells from normal and IPF lungs to discern disease-dependent changes within basal stem cells. Single-cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airways. Organoid and air-liquid interface cultures were used to investigate functional properties of basal cell subtypes. We found that basal cells included multipotent and secretory primed subsets in control adult lung tissue. Secretory primed basal cells include an overlapping molecular signature with basal cells obtained from the distal lung tissue of IPF lungs. We confirmed that NOTCH2 maintains undifferentiated basal cells and restricts basal-to-ciliated differentiation, and we present evidence that NOTCH3 functions to restrain secretory differentiation. Basal cells are dynamically regulated in disease and are specifically biased toward the expansion of the secretory primed basal cell subset in IPF. Modulation of basal cell plasticity may represent a relevant target for therapeutic intervention in IPF.
Topics: Aged; Alveolar Epithelial Cells; Basement Membrane; Case-Control Studies; Cell Plasticity; Cell Proliferation; Cell Self Renewal; Epithelial Cells; Female; Gene Expression Profiling; Humans; Idiopathic Pulmonary Fibrosis; Male; Middle Aged; RNA-Seq; Respiratory Mucosa; Single-Cell Analysis; Transcriptome; Young Adult
PubMed: 32692579
DOI: 10.1164/rccm.201904-0792OC -
The FEBS Journal Apr 2022The adenosine monophosphate-activated protein kinase (AMPK) is an integrative metabolic sensor that maintains energy balance at the cellular level and plays an important... (Review)
Review
The adenosine monophosphate-activated protein kinase (AMPK) is an integrative metabolic sensor that maintains energy balance at the cellular level and plays an important role in orchestrating intertissue metabolic signaling. AMPK regulates cell survival, metabolism, and cellular homeostasis basally as well as in response to various metabolic stresses. Studies so far show that the AMPK pathway is associated with neurodegeneration and CNS pathology, but the mechanisms involved remain unclear. AMPK dysregulation has been reported in neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and other neuropathies. AMPK activation appears to be both neuroprotective and pro-apoptotic, possibly dependent upon neural cell types, the nature of insults, and the intensity and duration of AMPK activation. While embryonic brain development in AMPK null mice appears to proceed normally without any overt structural abnormalities, our recent study confirmed the full impact of AMPK loss in the postnatal and aging brain. Our studies revealed that Ampk deletion in neurons increased basal neuronal excitability and reduced latency to seizure upon stimulation. Three major pathways, glycolysis, pentose phosphate shunt, and glycogen turnover, contribute to utilization of glucose in the brain. AMPK's regulation of aerobic glycolysis in astrocytic metabolism warrants further deliberation, particularly glycogen turnover and shuttling of glucose- and glycogen-derived lactate from astrocytes to neurons during activation. In this minireview, we focus on recent advances in AMPK and energy-sensing in the brain.
Topics: AMP-Activated Protein Kinases; Animals; Astrocytes; Brain; Energy Metabolism; Glucose; Glycogen; Mice
PubMed: 34355526
DOI: 10.1111/febs.16151 -
Cell Stem Cell Jan 2021The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we...
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1;TP63), we track and purify these cells as they first emerge as developmentally immature NKX2-1 lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1/TP63 cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
Topics: Cell Differentiation; Epithelial Cells; Humans; Induced Pluripotent Stem Cells; Lung; Pluripotent Stem Cells; Trachea
PubMed: 33098807
DOI: 10.1016/j.stem.2020.09.017 -
Cellular and Molecular Life Sciences :... Mar 2022There is a steadily growing interest in the use of mitochondria as therapeutic agents. The use of mitochondria derived from mesenchymal stem/stromal cells (MSCs) for... (Review)
Review
There is a steadily growing interest in the use of mitochondria as therapeutic agents. The use of mitochondria derived from mesenchymal stem/stromal cells (MSCs) for therapeutic purposes represents an innovative approach to treat many diseases (immune deregulation, inflammation-related disorders, wound healing, ischemic events, and aging) with an increasing amount of promising evidence, ranging from preclinical to clinical research. Furthermore, the eventual reversal, induced by the intercellular mitochondrial transfer, of the metabolic and pro-inflammatory profile, opens new avenues to the understanding of diseases' etiology, their relation to both systemic and local risk factors, and also leads to new therapeutic tools for the control of inflammatory and degenerative diseases. To this end, we illustrate in this review, the triggers and mechanisms behind the transfer of mitochondria employed by MSCs and the underlying benefits as well as the possible adverse effects of MSCs mitochondrial exchange. We relay the rationale and opportunities for the use of these organelles in the clinic as cell-based product.
Topics: Cell- and Tissue-Based Therapy; Humans; Lung Diseases; Macrophages; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mitochondria; Mitochondrial Dynamics; Paracrine Communication
PubMed: 35247083
DOI: 10.1007/s00018-022-04207-3 -
General anesthesia globally synchronizes activity selectively in layer 5 cortical pyramidal neurons.Neuron Jun 2022General anesthetics induce loss of consciousness, a global change in behavior. However, a corresponding global change in activity in the context of defined cortical cell...
General anesthetics induce loss of consciousness, a global change in behavior. However, a corresponding global change in activity in the context of defined cortical cell types has not been identified. Here, we show that spontaneous activity of mouse layer 5 pyramidal neurons, but of no other cortical cell type, becomes consistently synchronized in vivo by different general anesthetics. This heightened neuronal synchrony is aperiodic, present across large distances, and absent in cortical neurons presynaptic to layer 5 pyramidal neurons. During the transition to and from anesthesia, changes in synchrony in layer 5 coincide with the loss and recovery of consciousness. Activity within both apical and basal dendrites is synchronous, but only basal dendrites' activity is temporally locked to somatic activity. Given that layer 5 is a major cortical output, our results suggest that brain-wide synchrony in layer 5 pyramidal neurons may contribute to the loss of consciousness during general anesthesia.
Topics: Anesthesia, General; Anesthetics, General; Animals; Dendrites; Mice; Pyramidal Cells; Unconsciousness
PubMed: 35452606
DOI: 10.1016/j.neuron.2022.03.032 -
The Ocular Surface Jul 2021Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent...
PURPOSE
Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.
METHODS
Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs).
RESULTS
scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks.
CONCLUSIONS
Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.
Topics: Adult; Cell Differentiation; Cells, Cultured; Cornea; Epithelial Cells; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cells
PubMed: 33865984
DOI: 10.1016/j.jtos.2021.03.010 -
American Journal of Respiratory Cell... Mar 2023Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of...
Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function and in a repetitive bleomycin model . We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.
Topics: Animals; Mice; Bleomycin; Fibroblasts; Fibrosis; Idiopathic Pulmonary Fibrosis; Lung; Signal Transduction
PubMed: 36318668
DOI: 10.1165/rcmb.2022-0074OC