Respiratory Function Tests RFTs. Review Of Anatomy & physiology Lungs comprised of Airways Alveoli.
Cellular organization and biology of the respiratory system · • Respiratory bronchioles are...
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Endothelial cell ECM Elastin bundles Pulmonary blood circulation Bronchial circulation Acinus SMG Nerve Basal lamina Cartilage Pseudostratified epithelium Medium/blood substitute Air Lung epithelium Membrane Endothelial cells Side chambers • HBECs are seeded into ECM gels and generate spheres with basal, ciliated and secretory cells • AEC2s are seeded into ECM gels with fibroblasts and/or endothelial cells • In mouse-derived cultures, both AEC2s and AEC1s are present in these organoids; the efficiency of generating AEC1s from human AEC2s is currently limited Immune cells Dendritic cells Alveolar resident macrophages, interstitial macrophages Basal cells: TP63, KRT5, NGFR Function as multipotent stem cells Club cells: SCGB1A1, SCGB3A2 (data from mice) Immunomodulatory functions Goblet cells: MUC5AC, FOXA3, SPDEF Secrete mucins NE cells: ASCL1, CALCA Act as sensory cells; communicate with neurons Rare cells Ionocytes: FOXI1, CFTR high Tuft (brush ) cells: TRPM5, GNG13 Airways Ciliated cells: FOXJ1, β-tubulin IV Remove mucus out of the lung Alveoli AEC2s: SFTPC, DC-LAMP Stem cells; produce surfactant AEC1s: PDPN, AGER Large surface area; facilitate gas exchange Mesenchymal cells AEC2 associated fibroblasts (lipofibroblast-like): PDGFRA Peribronchial fibroblasts: GLI1, LGR6 Myofibroblasts: ACTA2 Smooth muscle cells: ACTA2, MYH11 Pericytes: PDGFRB Lymphoid cells (T and B cells) Basophils, eosinophils SMGs Acinar cells Serous: LTF, DCPP1 Mucous: MUC5B, TFF2 Myoepithelial cells: TP63, ACTA2 Multipotent stem cells; expel mucins out by contraction Human respiratory system Tissue organization and cell function Respiratory diseases Cystic fibrosis • Reduced airway surface fluid and thickened mucus inhibit mucociliary clearance and promote airway colonization by microbes • Risk factors: mutations (loss of function of CFTR) Asthma • Obstruction of small airways by goblet cell hyperplasia and excessive mucus production • Constriction of small airways and reduced air flow caused by hypersensitive smooth muscles and tissue fibrosis • Inflammation due to accumulation of eosinophils and other immune cells in airways • Risk factors: allergens, environmental pollutants, obesity, maternal–fetal exposure, family history Bronchopulmonary dysplasia • Delayed development of alveolar region • NE cell hyperplasia, inflammation • Risk factors: premature birth, maternal exposures Bronchiolitis • Inflammation in small conducting airways • Risk factors: viral infection, connective tissue disease, lung transplant rejection, burn-pit exposure, diacetyl exposure (rare) Chronic obstructive pulmonary disease Heterogeneous disorder including: Emphysema: • Abnormal, permanent enlargement of alveoli; reduced surface area for gas exchange • Loss of alveolar septa and reduced lung elasticity • Risk factors: smoking, mutations (loss of function of SERPINA) Chronic bronchitis: • Inflammation of mucosa in large airways, increased mucus production, cough • Risk factors: smoking, inhalational exposures, infection, gastroesophageal reflux Idiopathic pulmonary fibrosis • Progressive, irreversible replacement of alveoli with scar-like deposits of ECM, containing hyperplastic AEC2s, cysts of bronchiolar epithelium and inflammatory cells • Risk factors: advanced age, male sex, smoking, mutations (loss of function of telomerase genes (TERT, TR), surfactant proteins, and polymorphisms in MUC5B gene regulatory elements) Pulmonary arterial hypertension • Expansion of the tunica media in arterioles, endothelial plexiform lesions • Risk factors: mutations (BMPR2, ALK1), drug toxicity (fen-phen), HIV, connective tissue disease Lung cancer Major types of lung cancers include: ADCs (40% of cases): • Originate from AEC2s with five subtypes differing in morphology and clinical characteristics • Risk factors: mutations (gain of function: KRAS, EGFR; loss of function: CDK2A, KEAP1, STK11) SCCs (30% of cases): • Originate from basal cells and are characterized by stratified layers of flat, thin squamous cells • Risk factors: smoking, mutations (TP53, SOX3, TP63, PI3KCA, CDK2A, and FGFR1) SCLCs (15% of cases): • Originate from neuroendocrine cells and are one of the most aggressive cancer types in humans • Risk factors: smoking, mutations (RB1, TP53) Trachea Vocal folds Cartilage Respiratory bronchioles Bronchi Upper respiratory tract Pleura with lymphatic circulation Alveoli (~400 million) Distal bronchioles Bronchioles Intralobar bronchi Cell lineages Alveoli Self-renewal AEC2 AEC1 SMGs Self-renewal Mucous cell Serous cell Myoepithelial cell Basal cell Only after injury Only after injury • Use primary cells isolated from donor lungs after proteolytic digestion • Undifferentiated basal cells, known as HBEs, and AEC2s can be cultured in 2D and 3D modules • Used to identify growth and differentiation factors and screen for drugs to treat respiratory diseases • 2D cultures also allow measurement of transepithelial resistance (and hence, evaluate epithelial integrity) • 3D cultures are amenable for high-throughput screens • 2D and 3D cultures allow measurement of ion channel activity (e.g., CFTR) 2D cultures Air–liquid interface • HBECs are grown on transwell inserts in specialized cell culture medium • After reaching confluence, the liquid is removed to expose cells to air and the medium is changed to induce differentiation • By 2–4 weeks, pseudostratified epithelium is formed Pseudostratified epithelium Polyporous membrane Growth medium Lung on a chip • A microfluidic-based assembly in which lung epithelial cells are grown on one side of a membrane and stromal cells on the other surface • Liquid and air are circulated through the system to mimic air and blood flow in the lung 3D cultures (organoids) • Lung tidal volume is much smaller in mouse • Goblet cells are less abundant in mouse • SMGs are present only in upper-most trachea in mouse • NE cells are mostly solitary and scattered in human airways • Cartilage and basal cells are absent from mouse intralobar airways • Respiratory bronchioles are absent from mouse airways Airways Basal cell NE cell Ionocyte Tuft cell Ciliated cell Club cell Goblet cell Self-renewal Only after injury Mouse–human differences Lung cell culture models Cellular organization and biology of the respiratory system Brigid L. M. Hogan and Purushothama Rao Tata The lungs, which evolved in early terrestrial vertebrates, mediate the vital processes of supplying O 2 to the blood and removing CO 2 . Air enters through the mouth and trachea and is distributed through the lung lobes by a highly branched, tree-like system of tubes lined by pseudostratified epithelium. The intralobar airways supported by cartilage are known as bronchi; the smaller branches lacking cartilage are known as bronchioles. The bronchioles terminate in millions of tiny, thin-walled, highly vascularized sacs (alveoli) composed of specialized epithelial cells where gas exchange takes place. In addition to epithelial cells, the respiratory tract contains various mesenchymal cell types, including smooth muscle and fibroblasts that support epithelial homeostasis and lung functions as well as ectoderm-derived neurons. Although respiratory tract tissue shows limited turnover in homeostasis, damage to the respiratory epithelium can be repaired by resident tissue stem cells. Furthermore, immune cells within the lung help protect the lungs from infection and promote repair. Defects in the homeostatic and reparative mechanisms may lead to diseases such as chronic obstructive pulmonary disease, asthma, fibrosis and cancer, which together make a major contribution to mortality worldwide. STEMCELL Technologies PneumaCult™ is a serum- and bovine-pituitary extract (BPE)- free culture system that supports the expansion and differentiation of human airway epithelial cells, with the key feature being a pseudostratified epithelium consisting of goblet cells, basal cells, and motile cilia. PneumaCult™ -Ex Plus (#05040) • Long-term expansion medium for primary human airway epithelial cells. • Supports more rapid expansion and at least two additional passages with sustained differentiation potential compared to conventional expansion medium. • See the data at www.stemcell.com/ExPlus-data PneumaCult™ -ALI (#05001) • Differentiation medium for human airway epithelial cells cultured at the air–liquid interface (ALI). • Promotes extensive mucociliary differentiation with morphological and functional characteristics similar to the in vivo human airway. • See the data at www.stemcell.com/ALI-data Transwell® Inserts (#38023/#38024) • The recommended cultureware to use with PneumaCult™ products for optimal differentiation of airway epithelial cells at the ALI. Together, these products provide an optimized culture system for in vitro human airway modelling, supporting basic respiratory research, toxicity studies, and drug discovery studies. At STEMCELL, science is our foundation. We are Scientists Helping Scientists dedicated to making sure your research works. For more information, visit www.PneumaCult.com Abbreviations ADC, adenocarcinoma; AEC, airway epithelial cell; CFTR, cystic fibrosis transmembrane conductance regulator; ECM, extracellular matrix; EGF, epidermal growth factor; HBECs, human bronchial epithelial cells; NE, neuroendocrine; SCC, squamous cell carcinoma; SCLC, small-cell lung carcinoma; SMG, submucosal gland References 1. Weibel, E. R. Lung morphometry: the link between structure and function. Cell Tissue Res. 367, 413–426 (2017). 2. Hogan, B. L. M. et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 (2014). 3. Barkauskas, C. E. et al. Lung organoids: current uses and future promise. Development 144, 986–997 (2017). 4. Montoro, D. T. et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319–324 (2018). 5. Plasschaert, L. W. et al. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560, 377–381 (2018). 6. LungMAP - Available at: https://www.lungmap.net/ 7. Chakarov, S. et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science 363, eaau0964 (2019). 8. Okuda, K. et al. Localization of secretory mucins MUC5AC and MUC5B in normal/healthy human airways. Am. J. Respir. Crit. Care Med. 199, 715–727 (2019). 9. Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 3025–3036 (2013). 10. Desai, T. J., Brownfield, D. G. & Krasnow, M. A. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190–194 (2014). 11. Tata, A. et al. Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury. Cell Stem Cell 22, 668–683.e6 (2018). Acknowledgements We thank S. Randell and C. Barkauskas for valuable advice on the cellular composition of the human lung and human respiratory diseases, respectively. The poster content is peer reviewed, editorially independent and the sole responsibility of Springer Nature Limited. Edited by Christine Weber and Paulina Strzyz; copyedited by Lauren Beer; designed by Lauren Heslop. © 2019 Springer Nature Limited. All rights reserved. https://www.nature.com/articles/s41556-019-0357-7 Affiliations B.L.M.H. and P.R.T are at the Department of Cell Biology, Duke University Medical Center, Durham, NC, USA. Contact information: [email protected] or [email protected] Competing interests The authors declare no competing interests.
Transcript of Cellular organization and biology of the respiratory system · • Respiratory bronchioles are...
Endothelialcell
ECM
Elastinbundles
Pulmonaryblood circulation
Bronchialcirculation
Acinus
SMG
NerveBasal lamina
Cartilage
Pseudostratifiedepithelium
Medium/bloodsubstitute
AirLung epitheliumMembrane
Endothelial cells
Side chambers • HBECs are seeded into ECM gels and generate spheres with basal, ciliated and secretory cells
• AEC2s are seeded into ECM gels with fibroblasts and/or endothelial cells
• In mouse-derived cultures, both AEC2s and AEC1s are present in these organoids; the efficiency of generating AEC1s from human AEC2s is currently limited
Immune cells
Dendritic cells
Alveolar resident macrophages,interstitial macrophages
Basal cells: TP63, KRT5, NGFRFunction as multipotent stem cells
Club cells: SCGB1A1,SCGB3A2 (data from mice)Immunomodulatory functions
Goblet cells: MUC5AC, FOXA3, SPDEFSecrete mucins
NE cells: ASCL1, CALCAAct as sensory cells;communicate with neurons
Rare cellsIonocytes: FOXI1, CFTR highTuft (brush) cells: TRPM5, GNG13
Airways
Ciliated cells: FOXJ1, β-tubulin IVRemove mucus out of the lung
Alveoli
AEC2s: SFTPC, DC-LAMPStem cells; produce surfactant
AEC1s: PDPN, AGERLarge surface area; facilitategas exchange
Mesenchymal cells
AEC2 associated fibroblasts(lipofibroblast-like): PDGFRA
Peribronchial fibroblasts: GLI1, LGR6Myofibroblasts: ACTA2
Smooth muscle cells: ACTA2, MYH11
Pericytes: PDGFRB
Lymphoid cells (T and B cells)
Basophils, eosinophils
SMGs
Acinar cellsSerous: LTF, DCPP1Mucous: MUC5B, TFF2
Myoepithelial cells: TP63, ACTA2Multipotent stem cells; expel mucinsout by contraction
Human respiratory systemTissue organization and cell function Respiratory diseases
Cystic fibrosis• Reduced airway surface fluid and thickened
mucus inhibit mucociliary clearance and promote airway colonization by microbes
• Risk factors: mutations (loss of function of CFTR)
Asthma• Obstruction of small airways by goblet cell
hyperplasia and excessive mucus production
• Constriction of small airways and reduced air flow caused by hypersensitive smooth muscles and tissue fibrosis
• Inflammation due to accumulation of eosinophils and other immune cells in airways
• Risk factors: allergens, environmental pollutants, obesity, maternal–fetal exposure, family history
Bronchopulmonary dysplasia• Delayed development of alveolar region• NE cell hyperplasia, inflammation• Risk factors: premature birth,
maternal exposures
Bronchiolitis• Inflammation in small conducting airways• Risk factors: viral infection, connective
tissue disease, lung transplant rejection, burn-pit exposure, diacetyl exposure (rare)
Chronic obstructive pulmonary disease Heterogeneous disorder including:Emphysema:• Abnormal, permanent enlargement of
alveoli; reduced surface area for gas exchange
• Loss of alveolar septa and reduced lung elasticity
• Risk factors: smoking, mutations (loss of function of SERPINA)
Chronic bronchitis:• Inflammation of mucosa in large airways,
increased mucus production, cough• Risk factors: smoking, inhalational
exposures, infection, gastroesophageal reflux
Idiopathic pulmonary fibrosis• Progressive, irreversible replacement of
alveoli with scar-like deposits of ECM, containing hyperplastic AEC2s, cysts of bronchiolar epithelium and inflammatory cells
• Risk factors: advanced age, male sex, smoking, mutations (loss of function of telomerase genes (TERT, TR), surfactant proteins, and polymorphisms in MUC5B gene regulatory elements)
Pulmonary arterial hypertension• Expansion of the tunica media in arterioles,
endothelial plexiform lesions• Risk factors: mutations (BMPR2, ALK1),
drug toxicity (fen-phen), HIV, connective tissue disease
Lung cancer Major types of lung cancers include: ADCs (40% of cases):• Originate from AEC2s with five subtypes
differing in morphology and clinical characteristics
• Risk factors: mutations (gain of function: KRAS, EGFR; loss of function: CDK2A, KEAP1, STK11)
SCCs (30% of cases):• Originate from basal cells and are
characterized by stratified layers of flat, thin squamous cells
• Risk factors: smoking, mutations (TP53, SOX3, TP63, PI3KCA, CDK2A, and FGFR1)
SCLCs (15% of cases):• Originate from neuroendocrine cells and
are one of the most aggressive cancer types in humans
• Risk factors: smoking, mutations (RB1, TP53)
Trachea
Vocal folds
Cartilage
Respiratorybronchioles
Bronchi
Upperrespiratorytract
Pleurawith
lymphaticcirculation
Alveoli(~400 million)
Distalbronchioles
Bronchioles
Intralobarbronchi
Cell lineages
Alveoli
Self-renewal
AEC2 AEC1
SMGs
Self-renewal
Mucouscell
Serouscell
Myoepithelialcell
Basalcell
Onlyafterinjury
Onlyafterinjury
• Use primary cells isolated from donor lungs after proteolytic digestion
• Undifferentiated basal cells, known as HBEs, and AEC2s can be cultured in 2D and 3D modules
• Used to identify growth and differentiation factors and screen for drugs to treat respiratory diseases
• 2D cultures also allow measurement of transepithelial resistance (and hence, evaluate epithelial integrity)
• 3D cultures are amenable for high-throughput screens
• 2D and 3D cultures allow measurement of ion channel activity (e.g., CFTR)
2D cultures
Air–liquid interface• HBECs are grown on transwell inserts in
specialized cell culture medium • After reaching confluence, the liquid is
removed to expose cells to air and the medium is changed to induce differentiation
• By 2–4 weeks, pseudostratified epithelium is formed
Pseudostratifiedepithelium
Polyporousmembrane
Growthmedium
Lung on a chip• A microfluidic-based assembly
in which lung epithelial cells are grown on one side of a membrane and stromal cells on the other surface
• Liquid and air are circulated through the system to mimic air and blood flow in the lung
3D cultures (organoids)
• Lung tidal volume is much smaller in mouse• Goblet cells are less abundant in mouse• SMGs are present only in upper-most
trachea in mouse• NE cells are mostly solitary and scattered in
human airways• Cartilage and basal cells are absent from
mouse intralobar airways• Respiratory bronchioles are absent from
mouse airways
Airways
Basal cell
NEcell
Ionocyte Tuftcell
Ciliatedcell
Clubcell
Gobletcell
Self-renewalOnlyafterinjury
Mouse–human di�erences
Lung cell culture models
Cellular organization and biology ofthe respiratory system
Brigid L. M. Hogan and Purushothama Rao TataThe lungs, which evolved in early terrestrial vertebrates, mediate the vital processes of supplying O2 to the blood and removing CO2. Air enters through the mouth and trachea and is distributed through the lung lobes by a highly branched, tree-like system of tubes lined by pseudostratified epithelium. The intralobar airways supported by cartilage are known as bronchi; the smaller branches lacking cartilage are known as bronchioles. The bronchioles terminate in millions of tiny, thin-walled, highly vascularized sacs (alveoli) composed of specialized epithelial cells where gas exchange takes place. In addition to epithelial cells, the respiratory tract contains
various mesenchymal cell types, including smooth muscle and fibroblasts that support epithelial homeostasis and lung functions as well as ectoderm-derived neurons. Although respiratory tract tissue shows limited turnover in homeostasis, damage to the respiratory epithelium can be repaired by resident tissue stem cells. Furthermore, immune cells within the lung help protect the lungs from infection and promote repair. Defects in the homeostatic and reparative mechanisms may lead to diseases such as chronic obstructive pulmonary disease, asthma, fibrosis and cancer, which together make a major contribution to mortality worldwide.
STEMCELL Technologies
PneumaCult™ is a serum- and bovine-pituitary extract (BPE)-free culture system that supports the expansion and differentiation of human airway epithelial cells, with the key feature being a pseudostratified epithelium consisting of goblet cells, basal cells, and motile cilia.
PneumaCult™ -Ex Plus (#05040)• Long-term expansion medium for primary human airway
epithelial cells.
• Supports more rapid expansion and at least two additional passages with sustained differentiation potential compared to conventional expansion medium.
• See the data at www.stemcell.com/ExPlus-dataPneumaCult™ -ALI (#05001)• Differentiation medium for human airway epithelial cells
cultured at the air–liquid interface (ALI).• Promotes extensive mucociliary differentiation with
morphological and functional characteristics similar to the in vivo human airway.
• See the data at www.stemcell.com/ALI-data
Transwell® Inserts (#38023/#38024)• The recommended cultureware to use with PneumaCult™
products for optimal differentiation of airway epithelial cells at the ALI.
Together, these products provide an optimized culture system for in vitro human airway modelling, supporting basic respiratory research, toxicity studies, and drug discovery studies.
At STEMCELL, science is our foundation. We are Scientists Helping Scientists dedicated to making sure your research works.
For more information, visit www.PneumaCult.com
AbbreviationsADC, adenocarcinoma; AEC, airway epithelial cell; CFTR, cystic fibrosis transmembrane conductance regulator; ECM, extracellular matrix; EGF, epidermal growth factor; HBECs, human bronchial epithelial cells; NE, neuroendocrine; SCC, squamous cell carcinoma; SCLC, small-cell lung carcinoma; SMG, submucosal gland
References1. Weibel, E. R. Lung morphometry: the link between structure
and function. Cell Tissue Res. 367, 413–426 (2017).2. Hogan, B. L. M. et al. Repair and regeneration of the
respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 (2014).
3. Barkauskas, C. E. et al. Lung organoids: current uses and future promise. Development 144, 986–997 (2017).
4. Montoro, D. T. et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319–324 (2018).
5. Plasschaert, L. W. et al. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560, 377–381 (2018).
6. LungMAP - Available at: https://www.lungmap.net/ 7. Chakarov, S. et al. Two distinct interstitial macrophage
populations coexist across tissues in specific subtissular niches. Science 363, eaau0964 (2019).
8. Okuda, K. et al. Localization of secretory mucins MUC5AC and MUC5B in normal/healthy human airways. Am. J. Respir. Crit. Care Med. 199, 715–727 (2019).
9. Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 3025–3036 (2013).
10. Desai, T. J., Brownfield, D. G. & Krasnow, M. A. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190–194 (2014).
11. Tata, A. et al. Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury. Cell Stem Cell 22, 668–683.e6 (2018).
AcknowledgementsWe thank S. Randell and C. Barkauskas for valuable advice on the cellular composition of the human lung and human respiratory diseases, respectively.
The poster content is peer reviewed, editorially independent and the sole responsibility of Springer Nature Limited.Edited by Christine Weber and Paulina Strzyz; copyedited by Lauren Beer; designed by Lauren Heslop.© 2019 Springer Nature Limited. All rights reserved. https://www.nature.com/articles/s41556-019-0357-7
Affiliations B.L.M.H. and P.R.T are at the Department of Cell Biology, Duke University Medical Center, Durham, NC, USA.Contact information: [email protected] or [email protected]
Competing interestsThe authors declare no competing interests.
