BIOL463-Fall 2015
TEMPLATE FOR PROJECT OUTLINE – Due Oct 23
(please type!)
Student’s name: Nolan Shelley
Topic chosen: The Role of Basal Cells in Reconstituting Injured Alveolar Epithelial Cells in Mus musculus
SPECIFIC QUESTION: Do basal cells, which serve as progenitor cells for the conducting airway
epithelium, have the capacity to regenerate alveolar epithelial cells in response to different kinds of
lung injury?
HYPOTHESIS: Basal cells do have the ability to reconstitute alveolar epithelial cells following injury,
particularly in response to influenza infection.
EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):
- Lineage tracing has shown that Trp63+Krt14+ lung cells, which contain unique basal cell markers, can
emerge in distal lung alveoli, where they are not normally found, in response to severe influenza
injury – however, this experiment was highly flawed due to the initiation of lineage-tracing after
injury as opposed to before
- These cells were shown to generate alveolar cells expressing the AEC1 (alveolar epithelial cell type
1) marker gene Pdpn, though this gene is also expressed in some basal cells, calling into question
the legitimacy of this result
- Additionally, Trp63+ lung cells have been shown to accumulate in the bronchioles following
influenza infection, though there is little evidence for their presence in the bronchioles of normal
mice
- However, Krt14+ lung cells that underwent lineage tracing were not shown to generate AEC2s
(alveolar epithelial cell type 2), which are a critical epithelial lineage of alveoli
- Despite the flaws presented above, another experiment shows definitively that Scb1a1 + (which is a
marker of secretory club cells) cells in the bronchioles can give rise to AEC2s in response to alveolar
damage from bleomycin or influenza infection
o Because basal cells have been shown to give rise to such cells in the proximal airways, it
is possible that basal cells also give rise to these cells in response to such injury,
eventually leading to the regeneration of AEC2s, a hypothesis that has yet to be tested
PREDICTION(S):
- If the hypothesis is correct, then in response to influenza infection in mice I should be able to
observe basal cells give rise to alveolar epithelial cells (AEC1s and AEC2s) over time
- If the hypothesis is incorrect, then in response to influenza infection in mice I should not observe
any basal cells giving rise to alveolar epithelial cells. In contrast, I would expect a contribution
directly from Scgb1a1+ cells as shown in the experiment mentioned above, in addition to
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contributions from AEC2s, which have been shown to be progenitor cells of the alveoli during
normal adult homeostasis in mice
EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED
OUT SO FAR):
- Use lineage-tracing methods to determine whether basal cell descendents now exhibit alveolar
epithelial cell-like qualities
- This method will be elaborated on in the proposal
LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT
INFORMATION FROM EACH (this is the start of an annotated bibliography):
1.
Kotton, D.N. & Morrisey, E.E. Lung regeneration: mechanisms, applications and emerging stem
cell populations. Nature Medicine 20, 822-832 (2014).
This was the primary review article I used for my research. It outlines the pathways and progenitor
cells involved in lung development and regeneration in response to injury in the mouse. It describes
the in vivo research used to construct the models of the adult and developmental mouse lung,
including differentiation repertoires of stem cell candidates and the mechanisms of cell-lineage
labeling. It also goes into detail with regards to the mechanisms of epithelial regeneration in specific
regions of the lung, including the proximal and distal airways, the bronchoalveolar junction and the
alveoli. Lastly, it reviews the progress made in de novo regenerative therapies for the mouse,
including those that use induced pluripotent stem cells in vitro. It concludes with the current
therapeutic approaches being employed for human lung regeneration, such as the implantation of
stromal cells isolated from human bone marrow.
2.
Hogan, B.L. 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).
This was another review article used for developing my background knowledge on the topic of adult
lung regenerative mechanisms in vivo. It starts by providing a very detailed outline of the stages of
lung development in the embryo of the mouse, including branching morphogenesis and
alveologenesis. It then proceeds to outline the epithelial progenitor cell populations that mediate
adult lung homeostasis and regeneration in the various regions of the lung (just like the review
article above). It additionally provides comprehensive diagrams of the adult mouse bronchioles and
alveoli and their epithelial populations, which complimented the extensive written descriptions very
nicely. The article also describes the responses of the various regions of the adult mouse lung to
different injury-inducing treatments, including naphthalene and bleomycin. Lastly, like the above
article, it goes into the current methods for bioengineering lung tissue, including whole-lung
decellularization strategies.
3.
Rawlins, E.L. et al. The role of Scb1a1+ Clara cells in the long-term maintenance and repair of lung
airway, but not alveolar, epithelium. Cell Stem Cell 4, 525-534 (2009).
In this primary research article, the authors performed cell-lineage tracing on Scgb1a1-CreERTM ;
Rosa26R-eYFP transgenic mice to track the descendents of Scgb1a1+ (i.e. secretory) cells in the
BIOL463-Fall 2015
tracheal and distal airways, and alveoli over time. They analyzed the lungs of these mice during
postnatal growth, adult homeostasis, and in response to naphthalene- and hyperoxia-induced injury.
They found that while these cells give rise to secretory and ciliated cells in the distal airways during
postnatal growth and adult homeostasis, they do not give rise to alveolar epithelial cells. This was
also found to hold in response to the given lung injuries, despite previous conjectures that
bronchiolar epithelial cells contribute to alveoli during repair. The authors also found that Scgb1a1+
cells contribute only very minimally to epithelial repair in response to tracheal injury and during
postnatal growth, supporting the hypothesis that basal cells are the primary contributors to proximal
airway epithelial regeneration.
4.
Barkauskas, C.E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 30253036 (2013).
This article describes lineage-tracing experiments using Sftpc-CreERT2;Rosa26R-tdTm doubly
transgenic mice to determine the progenitor cells of the alveoli in the adult mouse lung during
maintenance and repair. They found that surfactant protein C-positive (Sftpc+) alveolar epithelial
type 2 cells (AEC2s) self renew and differentiate over about a year in the adult homeostatic lung,
additionally giving rise to AEC1s. Interestingly, authors found that the percentage of lineagelabeled Sftpc+ cells does not increase in response to bleomycin injury, suggesting that a Sftpc- cell
population helps to restore the AEC2 population in this instance, potentially including the Scgb1a1+
secretory club cells mentioned in the previous article. Additionally, they found that single lineagelabeled AEC2s grown in culture with Pdgfra+ lung stromal cells give rise to sphere-like colonies,
which they termed alveolospheres, containing both AEC2s and AEC1s.
5.
Kumar, P.A. et al. Distal airway stem cells yield alveoli in vitro and during lung regeneration
following H1N1 influenza infection. Cell 147, 525-538 (2011).
In this primary research article, the authors examine the role of basal cells of the proximal airways
in response to H1N1 influenza infection. They find that p63-expressing basal cells are intermingled
in the bronchiolar epithelium 11 days post-inoculation, and then fall in concentration by 21 days,
though they are not present at all in the bronchioles of normal mice. Additionally, basal cells were
also found in the damaged lung parenchyma, and formed discrete clusters of pods in the interstitial
lung (the area between the pulmonary alveoli and bloodstream). Then, they examined human
tracheal airway stem cells in vitro using pedigree tracking and found that they display significantly
robust differentiation into ciliated and mucin-producing goblet cells, far greater than that found in
distal airway stem cells. Furthermore, they found that mouse basal cells in vitro stain positive for
Aqp5, a marker of AEC1, in response to influenza infection. Lastly, using a poorly designed lineage
tracing experiment, they found that Krt5+ cells and their descendents migrated from the bronchioles
to local sites of interbronchiolar damage in response to influenza-induced injury.
6.
Kretzschmar, K. & Watt, F.M. Lineage tracing. Cell 148, 33-45 (2012).
This review article outlines the development of lineage tracing as a mechanism for tracking
descendents of specific progenitor cells. In particular, it delves into the use of genetic lineage
tracing in mice using the Cre-loxP system. It describes the general mechanism employed, involving
the placement of the Cre recombinase gene under the control of a lineage-specific promoter in one
transgenic line and the production of a reporter gene under the control of a ubiquitous promoter,
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flanked by a loxP-STOP-loxP sequence in a second transgenic line. In animals expressing both
constructs, Cre specifically activates the reporter in cells that express the lineage-specific promoter
by excising the STOP sequence. The article then elaborates on recent improvements in this system,
including temporal and spatial control of Cre activity through the use of the human estrogen
receptor and tamoxifen, as well as the advent of multicolor reporter constructs for lineage tracing
with two or more markers.
7.
Li, F. et al. Diversity of epithelial stem cell types in adult lung. Stem Cells International 2015,
728307 (2015).
This review article serves as a compliment to the first two references of this annotated bibliography,
providing an even more detailed look into the epithelial progenitor cells of the adult mouse lung. In
addition to discussing the usual stem cell candidates, such as basal cells in the proximal airway,
alveolar type II epithelial cells and naphthalene-resistant variant club cells within neuroepithelial
bodies of the distal airways, it also describes a subpopulation of as yet unidentified cells in the ducts
of the submucosal glands (in the proximal airway) and secretory cells in the bronchoalveolar duct
junction. It provides excellent illustrations of the various cell populations of mouse lung epithelia,
and a comprehensive description of potential niches for such progenitor cells, which I could not find
in other review articles. Lastly, it discusses lung cancer stem cells and the role of their niches in
supporting their capacity for self-renewal proliferation.
8.
Beers, M.F. & Morrisey E.E. The three R’s of lung health and disease: repair, remodeling and
regeneration. J. Clin. Invest. 121, 2065-2073 (2011).
This article aims not to provide an overly thorough overview of lung development, adult lung injury
or the pathways involved, but instead to elucidate similarities between what occurs during the
development process and injury response required to properly regenerate damaged cell lineages. It
outlines the possible avenues for future regenerative therapies, including the activation of local
progenitor populations, the insertion of exogenous lung progenitors and the promotion of local
proliferation of undamaged epithelium. Furthermore, it outlines the development of parenchymal
lung disease, in which the response stage can either result in appropriate repair, as is seen in injury
models of the mouse that were examined in the previous references, or in aberrant remodeling,
including excessive apoptosis and dysfunctional states of differentiation.
HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?
- Answering this question will expand upon our knowledge of the reparative mechanisms in the lungs
of adult mice in response to various forms of injury
- Knowledge of these mechanisms may eventually lead to the potential to regenerate human lung
tissue in response to serious injuries like chronic obstructive pulmonary disease (COPD) by
understanding the various progenitor cells involved and the signals that stimulate their
differentiation
- This serves as an alternative mechanism for injury repair, as opposed to de novo production of lung
tissue via stem-cell induction, which is another area of active research
BIOL463-Fall 2015
POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NONBIOLOGIST FAMILY AND FRIENDS):
- Through the understanding that models of injury and disease response in mice have remarkable
similarities to these processes in humans, the necessity of understanding the process of tissue
regeneration in the lungs of adult mice so as to provide potential avenues for disease treatment in
humans becomes very clear
ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:
- I guess everything is relatively well planned out except the annotated bibliography!
ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:
- A little late for this now!