lecture 25: stem cells in the lung

Question 1

From what are the tissues of the lung descendent?

Answer 1

• all three primary germ layers
• intrapulmonary nerves:
  
  • innervation of the developing lung is derived from neural crest progenitor cells of the ectoderm
• lung epithelium:
  
  • epithelial cells lining the lung are derived from progenitor cells of the foregut endoderm
• lung mesenchyme:
  
  • mesenchymal cells (smooth muscle, fibroblasts, pericytes, cartilage, etc) are derived from progenitor cells of the splanchnic mesoderm
• lung endothelium:
  
  • mesodermal mesenchymal progenitor cells responsible for vascularisation of the lung

Question 2

What is patterning of the foregut endoderm?

Answer 2

• the lung epithelium is formed by proliferation of an endodermal progenitor cell in the foregut endoderm
• the structure of the lung is then formed by formation of an epithelial lung bud surrounded by loosely packed mesenchymal cells
• branching morphogenesis results in the generation of the conducting and respiratory airways
• aveologenesis results in the formation of mature pulmonary gas-exchange units (alveoli)
• the lung epithelium arises through branching morphogenesis, which involves the proliferation and differentiation of lung epithelial stem cells located at the distal tip of the growing lung bud

Question 3

What has been shown with genetic tagging?

Answer 3

• by genetically tagging cells int eh foetal lung it has been show that Id2+ cells are the distal tip are able to give rise to all lung epithelial cells because the genetic marker (stained in blue) is passed on during cell division)

Question 4

What are lung embryonic stem cells?

Answer 4

• derived from the foregut endoderm
• multipotent epithelial stem cells capable of giving rise to all epithelial cell types in the lung

Question 5

From where is the lung mesenchyme derived?

Answer 5

• from the splanchnic mesoderm and gives rise to fibroblasts and smooth muscle in the developing lung

Question 6

What does FGF-10 mark?

Answer 6

• mesenchymal progenitor cells at the distal tip

Question 7

What regulates the proliferation and differentiation of lung epithelial stem cells?

Answer 7

• regulated by signals emanating from the mesenchymal progenitors
• progenitor cells at the distal tip → sit alongside and grow into the FGF-10 positive mesenchymal cells which then differentiate into the smooth muscle cells which then wrap around the airways and provide structure for the lung

Question 8

How complex is the adult lung?

Answer 8

• the adult lung is a complex organ comprised of at least 50 different cell types

Question 9

Is the composition of the lung uniform?

Answer 9

• the composition of the lung epithelium differs along the proximal-distal axis of the respiratory tree

Question 10

What happens following specific ablation of mouse airway epithelium?

Answer 10

• focal points of regeneration representing potential stem cell niches

Question 11

What does growth of lung epithelial stem/progenitor cells require?

Answer 11

• co-culture with lung mesenchymal stromal cells
• lung epithelial stem/progenitor cell = CD45^negCD31^negEpCAM^posCD24^low

Question 12

What stem cells are contained in the distal lung?

Answer 12

• the distal lung contains stem cells with multi-lineage (i.e. can differentiate into airway and alveoli epithelial lineages) and progenitor cells that are lineage restricted (i.e. generate only alveoli or airway cells)

Question 13

What is the hierarchical organisation of stem and progenitor cells in the adult lung?

Answer 13

• the hierarchical organisation of stem and progenitor cells in the adult lung differs along the proximal-distal axis of the respiratory with regional stem or progenitor cells being responsble for maintenance of different regions
• Multipotent stem cell
  
  • alveolar progenitor
    
    • AT2 cell
      
      • AT1 cell
  • airway progenitor
    
    • club cell
    • basal cell
      
      • SMG basal cell
        
        • mucous, ciliated, serous and duct cells
      • club cell
        
        • goblet cell
        • ciliated cell

Question 14

What are adult lung stem cells?

Answer 14

• in vitro clonogenic assay provides a powerful tool for the analysis of lung stem/progenitor cell organisation and regulation
• in vivo lineage-tracing techniques enable the identification of lineage hierarchies
• multipotent epithelial stem cells give rise to all epithelial lineages
• basal cells are airway progenitor cells which give rise to club cells, goblet cells and ciliated cells
• alveolar progenitors give rise to Alveolar type I and II cells
• submucosal gland basal cells submucosal gland cells and club cells, goblet cells and ciliated cells

Question 15

What will influence choice of a stem cell-based therapy for lung diseases and its likely therapeutic potential?

Answer 15

• vary across different disease
• acute
  
  • extent of injury
    
    • enough functional lung tissue remaining to participate in repair
      
      • endogenous or exogenous cellular therapy
    • not enough functional lung tissue to participate in repair
      
      • exogenous cell therapy
      • whole lung engineering
• chronic
  
  • timing of diagnosis
    
    • enough functiona…
      
      • type of disease/injury
        
        • genetic
          
          • exogenous cellular therapy
        • exposure
          
          • endogenous cell therapy
        • progressive
          
          • endogenous or exogenous cell therapy
        • viral/bacterial
          
          • endogenous or exogenous cell therapy
    • not enough …
      
      • whole lung engineering
      • exogenous cell therapy

Question 16

What are examples of lung diseases and their pathology, affected regions, and therapeutic target?

Answer 16

• Adult RDS
  
  • inflammation, hypoxaemia and impaired gas exchange
  • alveolar epithelium and capillary endothelium
  • regeneration of epithelia and endothelium
• asthma
  
  • inflammation, bronchospasm, and airflow obstruction
  • airway epithelium, myofibroblasts and airway smooth muscle
  • reduce inflammatory milieu, inhibit airway wall remodelling, inhibit smooth muscle hypertrophy and hyperplasia
• cystic fibrosis
  
  • CFTR mutation resulting in decreased mucociliary clearance and inflammation
  • airway epithelium
  • delivery of functional CFTR
• pulmonary emphysema (COPD)
  
  • loss of alveolar integrity and reduction of ventilation
  • alveolar epithelium, interstitial fibroblasts
  • generate alveolar septa and 3D alveolar structure
• pulmonary fibrosis
  
  • inflammation and fibrosis of alveolar tissue
  • alveolar epithelium, interstitial fibroblasts and endothelium
  • reduce inflammation, reduce alveolar epithelia loss and inhibit fibroblast proliferation
• what is the correct therapeutic target: the seed or the soil?

Question 17

What regulates the fate and specificty of stem cells?

Answer 17

• regulated by cues from the local microenvironment
• in the lung, mesenchymal cells form a niche for epithelial stem and progenitor
• intrinsic potential stem cell
• inductive microenvironment
  
  • permissive/supportive
  • restrictive/inhibitory

Question 18

correcting the activity of endogenous stem cells?

Answer 18

• in chronic respiratory diseases the injury and repair process goes wrong lead to:
  
  • A) lack of repair leading to degeneration
  • B) hyperproliferation of stem cells which can lead to hyperplasia, fibrosis and cancer
• development of drugs which switch on/off adult lung stem cells will offer the ability to correct the endogenous regeneration and repair process

Question 19

replacing stem cells through transplantation?

Answer 19

• the intrinsic potential of stem cells to self-renew and differentiate into specialised cells offers an alternative to whole tissue transplants to regenerate damaged tissues
• however, stem cells are regulated by their microenvironment, so stem cells transplanted into a “diseased” lung may not receive adequate signals for “healthy” lung regeneration
• if the microenvironment is damaged, can it be recondition to make it suitable for stem cell transplantation?

Question 20

For what do stem cells offer promise?

Answer 20

• as a vehicle for gene therapy of disease with single gene mutations like CF
• lung stem cells could be engineered with a correct copy of the cftr gene and transplanted back into patients to replace the “diseased” epithelium with “normal”

Question 21

Could non-viable lungs be used as scaffolds for recellularisation with stem cells to create bioengineered lungs as an alternative for lung transplants?

Answer 21

• maybe
• all cells removed leaving behind a matrix scaffold
• stem cells perfused back onto the matrix scaffold
• seeding stem cells onto decellularised trachea scaffolds has been successful in the clinic for replacement injured trachea

Question 22

What are key questions for lung stem cell therapies?

Answer 22

• what are the endogenous lung stem cells that maintain the lung throughout adult life?
  
  • stem cells during development are different to stem cells in the adult
• how do we isolate adult lung stem cells?
  
  • how pure a population do we need, how do we expand them?
• what are the key factors that regulate proliferation and differentiation of adult lung stem cells’?
  
  • how do we make sure the microenvironment is “conditioned” to promote repair and prevent uncontrolled proliferation?
• how do we drive ESCs or iPSCs to differentiate into lung cells?
  
  • how for do we need to differentiate them before transplantation?

Question 23

summary

Answer 23

• different lung diseases will require different stem cell therapies and the source of stem cells will depend on the therapeutic requirement (one size will not fit all!)
• the different sources of lung stem cells include (but are not limited too) embryonic stem cells, foetal lung distal tip stem cells, adult lung stem/progenitor cells and iPSCs
• the potential clinical uses of stem cells for respiratory diseases include:
  
  • regenerative medicine (cellular therapy and regulation of endogenous stem cells)
  • bioengineering (breathing new life into old lungs)
  • gene therapy (cystic fibrosis)