lecture 16: stem cell therapies for respiratory diseases

Question 1

How does the lung develop in the embryo?

Answer 1

• formation of blastocyst → gastrulation → ectoderm, endoderm, mesoderm
• most textbooks will say that the lung is of endoderm
  
  • true of the lung epithelium
• but lung is a very complex structure with contributions from other germ layers
• lung innervation → ectoderm
• mesoderm → lung endothelium, haematopoietic cells, lung mesenchyme

Question 2

Of how many cell types is the lung comprised?

Answer 2

• over 50 different cell types in the adult lung
• cartilage in the upper airways
• neural tissue
• epithelium itself made up of many different specialised epithelial cell types
  
  • ciliated cells
  • basal cells
  • club cells
  • dedicated type I and II pneumocytes in alveoli
• mesenchymal cells that are largely structural
• vascular
• endothelial
• resident haematopoietic regeneratation
• so when we talk about regeneration in the lung have to think of the stem cells that contribute to all of these different lineages

Question 3

What is patterning of the foregut endoderm?

Answer 3

• the lung epithelium is formed by proliferation of an endodermal progenitor cell in the foregut endoderm
  
  • foregut endoderm gives rise to thyroid, liver, pancreas, intestine and lung bud
  • lung bud branches out from this structure towards mesenchymal cells → interaction between the two kinds of cells
• 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)

Question 4

What are the stages of lung development?

Answer 4

• occurs over 38 weeks
• embryonic
  
  • bronchi
• pseudoglandular
  
  • bronchioles
  • terminal bronchioles
• canallicular
  
  • respiratory bronchioles
• saccular
  
  • alveolar ducts
• alveolar
  
  • alveolar sac
• continues on post birth

Question 5

What is epithelial branching morphogenesis?

Answer 5

• lung epithelial stem cells in the embryonic lung are located at the distal tip of the growing lung bud
• E11: just small bification where you just have developing out from the foregut endoderm
• very quickly go through a pattern of branching morphogenesis right to where have almost a fully developed embryonic lung at E16

Question 6

What is lineage tracing of lung embryonic stem cells?

Answer 6

• by genetically tagging lung embryonic stem cells by their expression of Id2 you can show that all lung epithelial cells are the progeny of Id2^pos stem cells because the genetic marker (blue) is passed on during cell division during development
• Id2 marks lung epithelial stem cells at the tip od the budding epithelium

Question 7

What is the epithelial hierarchy in the embryonic lung?

Answer 7

• lung embryonic stem cells, derived from the foregut endoderm, are multipotent epithelial stem cells capable of giving rise to all epithelial cell types in the lung
• endoderm
  
  • embryonic lung bud
    
    • lung embryonic stem cell (Id2+)
      
      • alveolar progenitor
        
        • alveolar type 2 cell
          
          • alveolar type 1 cell
      • bronchiolar progenitor
        
        • neuroendocrine cell
        • basal cell
          
          • club cell
            
            • ciliated cell
            • goblet cell

Question 8

What is the lung mesenchyme?

Answer 8

• mesenchymal cells derived from the splanchnic mesoderm give rise to fibroblasts and smooth muscle in the developing lung
• the developing lung epithelium is surrounded by mesenchymal cells
• differentiated smooth muscle cells surround the airways in the developing lung
• mesenchymal progenitor cells at the distal tip
• FGF-10 (blue) marks mesenchymal progenitor cells
• very close relationship between the epithelial and mesenchymal progenitors during development where the mesenchymal progenitors actually instruct the epithelial pgs what to do → reciprocal feedback → mesenchymal cells move up the side and develop into smooth muscle

Question 9

What is the crosstalk with the mesenchyme?

Answer 9

• the proliferation and differentiation of lung embryonic stem cells is regulated by signals emanating from the surrounding mesenchyme
• FGF-10 produced by mesenchymal cells promote epithelial growth
• epithelial progenitors grow towards the FGF-10 → proliferate → undergo branching morphogenesis → all of the epithelial cells of the lung
• mesenchymal cells begin to differentiate and wrap around → smooth muscles → lines the airways

Question 10

What is innervation of the lungs?

Answer 10

• innervation of the developing lung is derived from neural crest progenitor cells of the ectoderm

Question 11

What is vascularisation of the lung?

Answer 11

• vascularisation of the developing lung is derived from endothelial progenitor cells of the mesoderm
• flk-1 labels endothelial cells
• very complex
• you have a lot of endothelial cells wrapped around the alveoli and stretching up the airways
• very important for gas exchange
• in terms of understanding regenerative potential of the lung we need to know a lot more about how the endothelial progenitors contribute to vascularisation
• if the process goes wrong you have

Question 12

What is co-development of the heart and lung?

Answer 12

• new evidence that lung mesenchyme and endothelium shares a common ancestor with the cardiovascular system
• multipotent cardiopulmonary progenitor
  
  • cardiac inflow tract cardiomyocyte progenitor
    
    • atrial/sinus venosus myocardium
    • pulmonary vein myocardium
  • cardiopulmonary mesenchymal progenitor
    
    • pulmonary artery SMC
    • pulmonary vein SMC
    • airway SMC
    • pericyte
    • proximal vessel endothelium

Question 13

What is the most extensively researched stem cell hierarchy?

Answer 13

• hematopoeisis: a model stem cell hierarchy
• stem cells → progenitor cells → differentiated cells
• defining feature of stem cells is the capacy to self-renew and give rise to differentiated cells
• progressively become more restricted further and further down the heirarchy
• very well mapped out

Question 14

What is the cellular architecture of the adult lung?

Answer 14

• epithelium: lines upper airways, bronchioles, alveoli
• underneath a lot of different stromal cells
  
  • cartilage in upper airways
  • haematopoietic cells → interact bahlbehoigf

Question 15

What is flow cytometry for single cell analysis?

Answer 15

• adult stem cells are characterised by the expression of multiple markers which collectively specify the stem cell state and the lack of markers that are associated with differentiated cell lineages
• i.e. stem cells are often defined as Marker-A^pos Marker-B^pos Marker-C^pos
• the crucial advantage of using flow cytometry for stem cell research is the ‘single cell’ measuring principle: each and every cell is analysed as a single event → rare populations can be detected

Question 16

What was seen in clonal analysis of lung stem cells?

Answer 16

• growth of lung epithelial stem/progenitor in co-culture with lung stromal cells
• lung epithelial stem/progenitor cell = CD45^neg CD31^neg EpCAM^pos CD24^low
• plated with mesenchymal cells because they are required for lung development
• one particular population was able to form individual colonies

Question 17

What is multi-lineage differentiation?

Answer 17

• colonies derived from epithelial stem cells express (A) proteins and (B) genes associated with differentiated lung epithelial cells
• this shows that in the adult lung there are stem cells with multi-lineage potential (can differentiate into airway and alveoli epithelial lineages) and progenitors that are lineage restricted (generate only alveoli or airway cells)
• DAPI → indicator or airway cells
• SP-C → indicator of alveolar cells
• therefore the cell giving rise to the mixed colonies is likely the multipotent stem cell in the adult lung

Question 18

What is the lung epithelial stem/progenitor cell hierarchy as currently understood?

Answer 18

• multipotent epithelial stem cell
  
  • alveolar progenitor cell
    
    • alveolar epithelium
  • airway progenitor cell
    
    • basal cell
      
      • airway epithelium
    • SMG basal cell
      
      • submucosal glands
• airway progenitor gives rise to intermediate club cell → airway cell, ciliated cell
• still more work to be done
• but can say there is definitely a classical stem cell hierarchy in the lung, at least for the epithelium
• stem/progenitor cells have higher rates of proliferation compared to the more highly differentiated cells

Question 19

What is the lung epithelial stem cell niche?

Answer 19

• activity of stem cells is dictated or directed by the niche
• napthalene depletes club cells → treated mice
• distinct points of regeneration seen
• withing 3 days the airway is completely regenerated
• these points label with Cgrp → neuroepithelial body in the lung → possibility of a niche cell

Question 20

What is the microenvironmental regulation of stem cells?

Answer 20

• epithelial stem cell fate and lineage preferences determined by microenvironment
• stem cell has intrinsic potential to proliferate
• but directed by microenvironment
• microenvironment can be permissive/supportive or inhibitory/repressive
• need to think about the microenvironment when considering delivering stem cells → toxic microenvironment will prevent it from doing its job, need to have positive environment before it can do what it was planning to do

Question 21

What is the lineage specification of mesenchymal cells?

Answer 21

• only way you can get the epithelial stem cells to grow is by co-culturing them with mesenchymal cells
• similar to distal tip cells in that they produce a lot of FGF-10
• heterogeneous population
• populations of mesenchymal can be separated by flow cytometry
• have different potentials in terms of how they affect the progenitor cells
  
  • 166+ cells have very low proliferative capacity, don’t produce lots of lipofibroblasts, can still produce myofibroblasts, probably a more differentiated progenitor cell along the myofibroblast lineage
  • the other populations there is not a lot of difference
  • slightly more proliferation with the CD90+ population but both able to differentiate into the two types

Question 22

What are the epithelial-mesenchymal interactions?

Answer 22

• lung mesenchymal stromal cells interact directly with epithelial cells and create a niche for epithelial stem cells in culture
• when you culture the epithelial cells the mesenchymal cells surrounding it also differentiate
• signals eminating from the epithelium make the mesenchyme differentiate
• mesenchymal stromal cells producing FGF-10 results in proliferation of these cells
• still a lot of work to go in terms of understanding the different cues required for differentiation

Question 23

So what are lung stem cells?

Answer 23

• during development the epithelial component of the lung is derived from the endodermal layer which generates a population of lung embryonic stem cells that are capable of giving rise to all the epithelial lineages of the adult lung
• in the adult lung, the epithelium is maintained by the proliferation and differentiation of multi-lineage stem cells (can give rise to all epithelial lineages) as well as lineage-restricted progenitors (can give rise to only alveoli or airway epithelial cells)
• these adult lung epithelial stem progenitor cells can be isolated on the basis of the differential expression of EpCAM and CD24
• 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
• the lung is a complex organ made up of cells from each of the different germ layers, including epithelial, mesenchymal, endothelial, haematopoietic and neural cells → different stem cells are required to maintain each of the different components
• the fate and specificity of epithelial stem cells in the lung is determined by the interaction of all the different cell lineages in the lung which make up the local microenvironment
• stem cells are defined in context and the deregulation of the microenvironment can effect the homeostasis of epithelial stem cells and contribute to lung disease

Question 24

Why try and use stem cells as therapy for lung disease?

Answer 24

• lung diseases ranked 2nd in mortality, incidence, prevalence and cost
• by 2020 more than 1 in 6 deaths worldwide will be attributed to lung disease
• COPD is ranked 3rd in Global Burden of Disease
• asthma affects more than 300 million people worldwide
• cystic fibrosis is the most common autosomal recessive genetic disorder in caucasians affecting 1 in 1600 births
• annual economic burdern greater than 102 billion euro in Europe alone
• current therapies are essentially palliative and offer no prospect of cure or reversal of disease
• at best provide symptomatic relief
• many patients ultimately depend on lung transplantation

Question 25

What are some of the decisions that would have to be made to think about stem cells as a treatment for lung disease?

Answer 25

• lung disease/injury
  
  • acute
    
    • maybe not appropriate
  • chronic
    
    • option
• different diseases are going to take different approaches and possibly different stem cells/progenitor cells

Question 26

What are the different stem cells required for different diseases?

Answer 26

• Adult RDS
  
  • pathology: inflammation, hypoxemia, and impaired gas exchange
  • affected regions: alveolar epithelium and capillary endothelium
  • therapeutic target: regeneration of epithelia and endothelium
• Asthma
  
  • pathology: inflammation, bronchospasm and airflow obstruction
  • affected regions: airway epithelium, myofibroblasts and airway smooth muscle
  • therapeutic target: reduce inflammatory milieu, inhibit airway wall remodelling, inhibit smooth muscle hypertrophy and hyperplasia
  • i.e. reduce differentiation into mucus producing cells (targeting epithelial), but also want to be targeting mesenchymal cells to reduce differentiation into the myofibroblasts and smooth muscle
• Bronchiolitis obliterans:
  
  • pathology: inflammation and fibrosis of bronchioles
  • affected regions: airway epithelium
  • therapeutic target: regeneration of epithelia
• Bronchopulmonary dysplasia
  
  • pathology: necrotising bronchiolitis and alveolar septal injury
  • affected regions: alveolar epithelium, interstitial fibroblasts and capillary endothelium
  • therapeutic target: reduce inflamatory milieu, regeneration of alveolar septa and epithelium
• congenital lung hypoplasia
  
  • pathology: incomplete development of lung resulting in reduced number or size of bronchopulmonary segments or alveoli
  • affected regions: alveolar epithelium, interstitial fibroblasts and capillar endothelium
  • therapeutic target: generate alveolar septa and 3D alveolar structure
  • lung is underdeveloped so need to regenerate everything
• cystic fibrosis
  
  • pathology: CFTR mutation resulting in decreased mucociliary clearance and inflammation
  • affected regions: airway epithelium
  • therapeutic target: delivery of functional CFTR - can you use stem cells to do this?
• neonatal RDS
  
  • pathology: insufficient surfactant production in the lungs
  • affected regions: alveolar epithelium and capillary endothelium
  • therapeutic target: generation of surfactant production and regenerate epithelia and endothelia
• pulmonary emphysema (COPD)
  
  • pathology: loss of alveolar integrity and reduction of ventilation
  • affected regions: alveolar epithelium, interstitial fibroblasts and capillary endothelium
  • therapeutic target: generate alveolar septa and 3D alveolar structure
• pulmonary fibrosis:
  
  • pathology: inflammation and fibrosis of alveolar tissue
  • affected regions: alveolar epithelium, interstitial fibroblasts and endothelium
  • therapeutic target: reduce inflammation, reduce alveolar epithelia loss and inhibit fibroblast proliferation
• sarcoidosis
  
  • pathology: inflammation accompanied by granuloma formation
  • affected regions: epithelium
  • therapeutic target: reduction of inflammation and regeneration of epithelia

Question 27

What is targeting of endogenous stem cells?

Answer 27

• in chronic respiratory diseases the injury and repair process goes wrong leading to:
  
  • lack of repair, or
  • too much proliferation of endogenous stem cells → hyperplasia 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
• challenges:
  
  • in some cases the intrinsic potential of stem cells may be affected by the disease, while in other cases the microenvironment may be damaged
  • do we target the seed (stem cell), the soil (microenvironment) or both?

Question 28

What are stem cell transplants in terms of lung regeneration?

Answer 28

• 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
• challenges:
  
  • what is the best source of stem cells (embryonic or IPS-derived or adult)?
  • lung epithelial stem cells delivered to a “diseased” lung may not receive adequate signals for “healthy” lung regeneration
  • if the microenvironment is also damaged, the fate and specificity of the exogenous stem cells may be altered
  • how can the microenvironment be reconditioned to make it suitable for stem cell transplantation?

Question 29

What are potential sources of stem cells for therapy?

Answer 29

• embryonic stem cells
  
  • can’t use them explicitly because of risk of teratoma formation
  • need to encourage them to undergo differentiation
• induced pluripotent stem cells
  
  • transfection of somatic cells with factors to induce pluripotency → iPSC → endoderm → transplanted
  • need to make sure that any modifications to persons cells are safe
• adult stem cells
  
  • safest approach
  • proliferative potential and ability to expand these cells is far less
• more work needs to be done in examining safety and efficacy of those different types

Question 30

How can stem cells be used as a vehicle for gene therapy?

Answer 30

• expansion of stem cells in vitro provides an opportunity to correct the genotype of patients with monogeneic disorders such as cystic fibrosis
• 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” epithelial cells
• challenges:
  
  • engraftment of stem cells into the lung epithelium is often very poor and will require successful depletion of the “diseased” epithelium without harming the microenvironment

Question 31

How can stem cells be used in bioengineering?

Answer 31

• decellularisation is a technique in which the lung is depleted of cellular material to generate a matrix scaffold that can be re-seeded with stem cells to generate a bioengineered lung that can be transplanted back into patients
• only put back the epithelial cells
• would need to understand proliferative and differentiative potential of all the different cells
• seeding stem cells onto decellularised trachea scaffolds has been successful in the clinic for replacement of injured trachea → important to check that ciliated cells are beating in the right direction

Question 32

What are key questions for lung stem cell therapies?

Answer 32

• what are the endogenous lung stem cells that maintain the lung throughout adult life?
• how do we isolate adult lung stem cells?
• what are the key factors that regulate proliferation and differentiation of adult lung stem cells?
• how do we drive embryonic stem cells or iPS cells to differentiate into lung cells?

Question 33

summary

Answer 33

• 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 to) embryonic stem cells, foetal lung distal tip stem cells, adult lung stem/progenitor cells and induced pluripotent stem cells
• 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)
  • immunomodulation (mesenchymal stem cells)