Lung Development, Lung Stem Cells

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Lung Development, Lung Stem Cells and Lung
Disease
Jay Rajagopal 9 July 2007
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Questions

• How are organs made in the embryo?
• How can the principles of developmental and stem
  cell biology can be applied to understand organ
  biology in the adult?
• How can the tools of developmental and stem cell
  biology can be used to study human disease?

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Developmental Biology of the Lung

• Lung Organogenesis
• Branching morphogenesis
• New approaches to study development of organs
• Lung Stem Cells
• Organ maintenance
• In Disease
• As therapy
• Cystic Fibrosis
• Lung cancer (Metaplasia)

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Human embryo, 4 weeks after fertilization


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How are organs specified?
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Schematic Lung Anatomy
Trachea
PB
b
Alv
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Development of the Lung 2 phases First
Production of the conducting airways
Second Evolution of the tissue required for gas
exchange
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Mammalian Lung Development
Bud Formation
Groove Formation
Closure
Branching Begins
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How can we study lung development at a molecular
level? Genetic regulation of a gene specific
for the lung Which gene? Surfactant proteins

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Surfactant Protein
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Transcriptional Control of the Developing Lung
Analyze of the control of the Surfactant gene
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Gene control by transcription factors
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Nkx2.1 gene Nkx2.1 Knockout mice No
distal alveolar, bronchiolar, or mesenchymal lung
structures
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Parsing of the Endoderm identification of
Nkx2.1
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Branching Morphogenesis
How do we build organs?
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Classic Histologic Description of Lung
Development
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Pseudoglandular Phase Air passage form
resembling tubes Most of the main bronchial tree
is formed
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Canalicular Close association of the
capillaries and epithelial lumen Thinning of the
mesenchyme
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Saccular Phase 0.3 micron barrier forms Type I
cells and Type 2 cells
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Formation of alveoli (Postnatal) Septation
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Classic examples of embryologic defects
associated with abnormal Lung development Bronc
hogenic Cysts (Incidence unknown) Tracheoesophag
eal fistula (1/2000-4000 live births)
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Congenital Diaphragmatic Hernia
1/3000 Live births
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• Can other model systems teach us about
• human lung development?

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Drosophila respiratory organ
Could a similar mechanism govern the formation of
the Fly trachea and the human lung?
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Easy to screen for mutations in Drosophila
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Branch formation in the respiratory tree
FGF clusters of cells dictate where a branch
will form Breathless tracheal cells FGF-R
(Tyrosine Kinase) Sprouty Promotes branches by
inhibiting FGF
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Branching Morphogenesis

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Complex interaction of signaling molecules
organizes branching
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Identification of New Genes Responsible for
lung Development - 1250 Lung Genes so far
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Lung bud screen
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Endoderm Distal Tip
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Endoderm Distal Tip
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Endoderm Airways
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Endoderm ciliated cells
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Mesoderm Mesenchyme
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Mesoderm Vascular
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Capillaries
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The Great Vessels of the Lung
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Cartilage
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E14.5 Unique Transcriptional Domains
Punctate Peri Endodermal (Vascular)
Mesenchyme Sheath excluding Tip (SM)
Airway
Tip
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In Vitro Lung Culture
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(No Transcript)
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Assays of gene function
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Viral gene expression in infected cultured lung
buds
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Stem Cells
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2 Kinds of Stem Cells

• Embryonic

• Adult

• most primitive
• can form all cell types
• immortal in culture
• plentiful

• organ specific
• can form few cell types
• limited lifespan
• hard to isolate

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Embryonic Stem Cells
Smith (2001) Ann. Rev. Cell. Dev. Biol. 17
435-462
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Stem Cells
self-renewal
differentiated cell
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Stem Cells and Regeneration in the Lung

• Proximal Epithelium
• Distal Epithelium

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Mature Lung
Basal Cells Ciliated Cells Glands Goblet Cells
Neuroendocrine Ciliated Cells Clara Cells
Neuroendocrine Goblet Cells Type 1 Cells Type
2 Cells
Trachea
Airway
Alveolus
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Stem Cells and Regeneration in the Lung

• Proximal Stem Cell
• Distal Stem Cell

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Tracheal Epithelium
E-Cadherin
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Tracheal Cells
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Label retaining cells

• How fast do stem cell replicate?
• Paradoxically stem cells replicate slowly
• How do we find these cells?

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Label Retention Cells in the Skin
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Label retaining cells are present in tracheal
epithelium
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Cell therapy?

• We can now attempt to culture these cells,
  differentiate them, and even transplant them

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Diseases of the Lung

• Associated with premature death
• Shortness of Breath
• Limited treatment options
• Few if any curative therapies

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Lung Disease

• COPD
• Emphysema
• Bronchitis
• Lung Cancer
• Asthma

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The Importance of Lung Diseases

• 1 in 7 American deaths are due to lung disease
• Lung disease is in the aggregate the 3rd leading
  cause of death

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Cystic Fibrosis

• A single point mutation in an ion channel.
• Causes secretion of a thickened mucus from the
  glands of the trachea and lung, the pancreas, and
  other secretory structures.
• Principal complication is airway blockage due to
  dehydrated mucous entering the lung and making it
  hard to breathe.
• The mucus traps bacteria causing persistent
  unclearable infection that eats away the lung.

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(No Transcript)
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Gross Pathology
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Cystic Fibrosis History

• 1949 Lowe postulated the existence of a single
  gene - autosomal recessive inheritance of
  pancreatic fibrosis
• 1986, Quinton, High levels of salt in the sweat
  of patients with CF, established sweat ducts are
  impermeant to Cl-
• 1986-1989, Patch Clamping of nasal and airway
  epithelium
• 1989, Cloning of the gene - CFTR

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Status of therapy for Cystic Fibrosis

• Patients now often live beyond the third decade
• Most progress has been associated with
  improvements in supportive care
• Gene therapy efforts failed

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Cystic Fibrosis

• Chronic, very debilitating and ultimately lethal
  disease
• Affects young people
• Represents a obvious hurdle for molecular
• medicine
• Target for regenerative medicine

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Cystic Fibrosis as a model for a Cell Therapy

• Well defined simple proximate cause of disease
• Airways exhibit surprisingly robust regeneration

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Basis for a curative therapy for CF

• Cure to Cystic Fibrosis will be rely upon
  understanding airway regeneration
• Isolation and characterization of airway
  progenitor cells
• Gene therapy should work, but must be
  accompanied by prospective ID of a stem or
  progenitor cell.

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Model of Tracheal Regeneration
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Tracheal Epithelium
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Tracheal Transplant Histology
Time 0 days
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Transplant Time Course Day 2
Recipient Donor
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Transplant Time Course Day 3
Recipient Donor
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Transplant Time Course Day 7
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Ck14 population expands during regeneration
Donor, Day 7
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Lung Cancer
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(No Transcript)
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Where does lung cancer come from?
What is the cell of origin of lung cancer?
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Cancer Stem Cells

• Do we need to target all cells in a cancer or
• Should we just try to kill the stem cells?

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Metaplasia
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What about Asthma?
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Smooth Muscle Cell Replication
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Stem Cells and Regeneration in the Lung

• Proximal Epithelium
• \
• Distal Epithelium

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Thanks