Nihal Alam 5/1/20

Current Standing of Creating a Functional Lung

Through Stem Cell Research

The topic of focus was looking at current search regarding the creation of functional
lungs through stem cell research. Throughout the world, getting a necessary lung transplant for a
dire lung condition is becoming urgent as millions of people die. Therefore, the articles being
investigated are:

“Generation of functional lungs via conditional blastocyst complementation using pluripotent

stem cell” by Mori, Furhashi, Danielsson, Hirata, Kakiuchi, Lin, Ohta, Riccio, Takahashi, Xu,
Emala, Lu, Nakauchi and Cardoso at the Columbia University Irving Medical Center
“Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized
whole lung scaffolds” by Wagner, Bonvillain, Jensen, Girard, Bunnell, Finck, Hoffman and
Weiss at the Department of Pharmacology at Tulane University School of Medicine.
“Regeneration of the lung: Lung stem cells and the development of lung mimicking devices” by
Schliders, Eenjes, Riet, Poot, Stamatilais, Truckenmuller, Hiemstra and Rottier at the Department
of Pediatric Surgery, Erasmus, Medical Center Sophia’s Children Hospital.

Stem cells can be directed to create a specific type of cell of choice. In the first research
paper1, pluripotent stem cells are the best choice. Pluripotent stem cells (PSC) can differentiate
into a number of different cells which from any of the three germ layers. Here, they explored
creating lungs in animals from donor stems cells by using a blastocyst complementation
approach (which is when you inject stem cells into blastocyst stage of an embryo). After, PSC
must reach its organ niche and respond to specific signals the organogenesis program gives off.
To avoid problems of lung agenesis(failure of organ to develop), they used a gene-ablation
strategy at a specific promoter where this blastocyte could be helped in thriving1. Injection of
VPA(Valproic acid which is an histone deacetylase inhibitor and this means that it blocks
enzymes that remove the acetyl group from histone proteins that make DNA less accessible to go

1
(Mori et al., 2019)
through transcription process)/LIF-treated (Leukemia inhibitory factor which stops
differentiation of cell) was put into the host and it was found that they had functional lungs.1
After more was tested by injection of a2i/VPA/LIF which made the lungs come out to be more
highly efficient.1 This is due to chromatin modifications in DNA (Deoxyribonucleic acid ) and
methylation (adding a methyl group to DNA which blocks a certain region from getting
transcribed) of PSC pluripotency. For these series of experiments, most changes were done at the
molecular level to understand how the genes get involved. For example, after treatment they also
analyzed differences in patterns of histone (protein that wraps around DNA) modifications
between different treatments. After they did a test using CBC (Conditional Blastocyst
Complementation which means that it leaves a place in the host that allows start of
organogenesis by PSC) approach to see whether the enhanced developmental potential of two
factors help generate lungs to fully functional throughout their life.1 In the model it showed the
chimeric animals with lung completion normally to adulthood. To summarize, a gene-ablation
(meaning method of modifying DNA to hinder production of a specific gene) strategy was used
to get a niche for CBC with something enhancing PSC for helping the developmental potential of
the lungs. This approach takes advantage of tissue interactions in vivo to help overcome
generating lung and trachea in host embryos. It took complementation of defective respiratory
progenitor niches to make the formation of a functional lung.1 However, the downside is that
there are still challenges for future use of this technology in the clinical setting and therefore
more understanding of immune tolerance and what happens more on the molecular level on
humans.
Moving on, the next paper2 talks about the option of how an ex vivo (outside cell)
approach might work through bioengineering. Various types of cells were looked at for
recellularization (which is when Extra Cellular Matrix (group of proteins and such that support to
surrounding cells) of tissues or organs are trying to re-create the function specific to the organ) of
acellular (life that exists without a structure) lung scaffolds (temporary structure). The candidates
for this were whole lung cell suspensions, endogenous progenitor cells, embryonic and adult
stem cells and induced pluripotent cells to see if they can repopulate acellular matrices (plays
role in regenerating tissues or organs). Decellularization and recellularization procedures are
looked at in both theoretical and practical sense and seeing for potential immune responses in the

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(Wagner et al., 2013)
scaffolds.2 For organ scaffolds to be created, removal of cell population needs to be done while
making sure no change is done to the physical characteristics of the structure.3 Decellularization
techniques are explained and most common approach is use of detergents which include Triton
X100, SDC and more. Optimal decellularization is probably complete absence of visible cellular
or nuclear material. Retention of key ECM components is very necessary. There are a lot of
structural and functional molecules of the lung like collagens, elastin and more that need to be
preserved and highly conserved.2 Through these procedures, it is suggested that the proteins are
not at all removed. Each of the candidates mentioned above were explored and the benefits were
noted. Among post-natal cells, autologous adult cells, embryonic stem cells and induced
pluripotent cells, lung epithelial progenitor cells, lung-origin mesenchymal stem cells, it seems
the induced pluripotent cells make most sense. It is said for all others that though they have their
advantages, some are not practical and most are just not known enough about to understand
implications. However, though what already stated above seems hard to accomplish, the
recellularization is harder as niches are to be characterized for stem-progenitors to be in the lung
or else the specific parts of the lungs cannot be formed or distinguished. More studies were
shown about having implantation of recellularized scaffolds in rats but the lungs did not maintain
proper oxygenation and so it is not able to be used at all clinically. In the end though there were
developments, there still are challenges in developing functional lung tissues in an ex vivo
matter.
Finally, in the last paper,3 it is discussed how the lung can be regenerated and how stem
cells can aid in development of this. It is first described how different stem cells or progenitor
cells reside in the lung and then how plasticity (ability to be changed or shaped, changed ) of
different cell types upon injury make it activate its repair models4 and the last part of the paper
won’t be discussed because it is not really relevant to stem cells. To start off it is first discussed
the potential use of epithelial stem cells of the lung. Since it is known that the lungs of humans
are lined by pseudostratified epithelium which are made up of basal cells, the progenitor cells
can be placed here. In order for it to differentiate into cells usable for the lung, it becomes a Krt8
positive luminal precursor and then differentiates.5Neuroendocrine cells and Scgb1a1+ secretory
cells are also self-renewing and this gives rise to ciliated cells. The way to accomplish this

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(Wagner et al., 2013)
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(Schilders et al., 2016)
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(Schilders et al., 2016)
differentiating is “upon depletion of luminal cells by So2 exposure, basal cells proliferate and
subdivide into two populations, N2ICD and c-myb positive, differentiating into secretory and
ciliated cells.3 After the loss of these basal cells, secretory cells differentiate into functional
progenitor basal stem cells which then further differentiate into things of choice. To summarize,
Basal Stem Cells go into basal luminal precursor cells and there are three of them (Kr5+,
Trp63+, Krt8+) which then go into either goblet, club, ciliated or neuroendocrine cells. For the
repair side, basal stem cell has two sides which it can go through. The first side goes into
N2ICD+ which is a cuboidal form into a more circular form and then into a club cell which can
be formed into either a goblet or ciliated cell. The other pathway is it becoming a c-myb+ as
cuboidal then changing shape to oval and then into a ciliated cell which can also become a
neuroendocrine cell. After, a look is taken at regeneration of distal and alveolar airway
epithelium after injury. In here a variant club cell and neuroendocrine cell can be ordered to
differentiate into different alveolar cells (which includes alveolar type 2, alveolar type 1, alveolar
progenitor cell). Other epithelial progenitor cells that were talked about is a subset of Scgb1a1
+ which co-express the AT-II marker surfactant protein which helps differentiate in bronchiolar
and alveolar lineages in vitro. 3 These cells repair itself after certain injuries that happen to the
lung for example for this case it would be after influenza or bleomycin-induced injury. The lungs
cells have different plasticity. Basal cells can dedifferentiate into ciliated or secretory cells. 3 The
Hippo Pathway is very effective for it to require for dedifferentiation of secretory cells and so it
is important in stimulating the regeneration of pseudostratified epithelium. This is the general
background information to understand the last topic being discussed which is the regenerative
medicine section. There are different pathways of signaling for “maintaining a quiescent
homeostatic or inducing a proliferating regenerating epithelium”3. The signaling here consists of
a cross-talk and feedback loops between epithelial cells but also between epithelial and
mesenchymal cells. Hh signaling for example signals between stimulating proliferation and
quiescence and is active to maintain quiescence however upon injury Hh signaling is inhibited to
stimulate epithelial proliferation. 3Other pathways were discussed but the main idea here was that
though therapies are not yet available, inducing or inhibiting pathways by activation of
endogenous lung progenitor cells3 will prove to be very beneficial.
Things learned for in vivo is that it is indeed feasible to create a functioning lung if the
right inhibitors and factors are injected for it to interact with the genes in order to get a good
response. The only down side is, the side of one’s immune reaction cannot be determined. For
the ex vivo part, it was learned that only the basics were really available for some progress in
creating a lung (basics referring to not fully functional like seen in first research paper). The
process to create a lung is using organ scaffolds and having candidates (which can be a host of
different stem cells) ready for it to host the process. Depending on the process of decellularizing,
the desired outcome may not come to be and the recellularization process will be affected leading
in a lung that is not as good.
The reason for interest in this topic is because of cool and interesting the possibility is of
creating an exact copy of a lung without requiring a donor body. Also, because this is a relatively
new field which people are exploring in. Lastly due to family being affected, it really sparked a
curiosity to understand what options are available and to see what research is out there that might
help.
The societal implications can be very significant if the final stage of this research is
complete. Due to the shortage of lungs for donation to those in the last stages of pulmonary
failure, a lot of people end up dying. However, if this research were to be created, then this could
be used to save millions of people’s lives as transplants could now be possible since these stem
cells will be from the patient. Furthermore, even if there are lungs available, the patient may not
be suitable for it and end up rejecting the lung all together. Since this lung is being manufactured
from your own cells, your body has a barely a chance of rejecting it. The patient outcome would
definitely be a lot higher for those with lung transplants. Lastly, if there were multiple ways to
bring about the creation of lungs through stem-cells (ex vivo or in vitro) then a developmental
therapy for bringing back one’s lung can happen or directly have a lung created from your own
cells and be operated on.
A future experiment that could be explored is trying to have the natural development of
the patient’s lung through stem cells from his body happen outside in a controlled environment.
This may seem safer as an in vitro approach can maybe mess up along the cellular pathway while
development. However, during the procedure of this process, a faster way to decellularize and
recellularization things must be found out which can help serve in urgent, time strained situations
for getting someone a lung transplant as fast as possible. Another future experimental idea could
possibly be looking at the GAG protein (structural and functional molecule in ECM) and see
what is retained after decellularization which may help scientists understand if this protein is
responsible for triggering any immune responses. In addition, for the in vitro approach,
understanding histocompatibility barriers (looking more into the histones of the DNA and how
they are impacted) in relation to immune tolerance.
References
Mori, M.; Furuhashi, K.; Danielsson, J. A.; Hirata, Y.; Kakiuchi, M.; Lin, C.-S.; Ohta, M.;
Riccio, P.; Takahashi, Y.; Xu, X.; Emala, C. W.; Lu, C.; Nakauchi, H.; Cardoso, W. V.
Generation of Functional Lungs via Conditional Blastocyst Complementation Using
Pluripotent Stem Cells. Nature Medicine 2019, 25 (11), 1691–1698.
Wagner, D. E.; Bonvillain, R. W.; Jensen, T.; Girard, E. D.; Bunnell, B. A.; Finck, C. M.;
Hoffman, A. M.; Weiss, D. J. Can Stem Cells Be Used to Generate New Lungs?Ex
Vivolung Bioengineering with Decellularized Whole Lung Scaffolds. Respirology 2013, 18
(6), 895–911.

Schilders, K. A. A.; Eenjes, E.; Riet, S. V.; Poot, A. A.; Stamatialis, D.; Truckenmüller, R.;
Hiemstra, P. S.; Rottier, R. J. Regeneration of the Lung: Lung Stem Cells and the
Development of Lung Mimicking Devices. Respiratory Research 2016, 17 (1).