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European Biopharmaceutical Review
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Stem cells are unique from all other bodily cells thanks to three
traits: firstly, they are capable of dividing and renewing themselves
for long periods, unlike muscle or nerve cells; secondly, they are
unspecialised; and finally, they can differentiate into specialised
cells (1).
Scientists are trying to understand two fundamental properties in relation to their long-term self-renewal:
- Why
can embryonic stem cells proliferate indefinitely in the laboratory
without differentiating, when most adult stem cells cannot?
- What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?
Study Interests
Adult
stem cells – also referred to as somatic stem cells – have been
identified in many organs and tissues, including the brain, bone marrow,
peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart,
gut, liver, ovarian epithelium and testis. They are believed to reside
in specific areas of each tissue in the so-called ‘stem cell niche’ and
can remain quiescent for long periods of time until they are activated
to generate more cells to maintain normal tissues, or cells lost due to
disease or injury.
Researchers are hoping to identify how
undifferentiated stem cells can become differentiated cells in
developing tissues and organs, as some of the most serious medical
conditions – such as cancer and birth defects – are due to abnormal cell
division and impaired differentiation. Particularly important are
transcription factors: proteins that directly or indirectly interact
with DNA, thereby switching genes on or off during embryo development.
For instance, Oct-4 and Nanog are two crucial transcription factors
associated with maintaining pluripotent stem cells in an
undifferentiated state, capable of self-renewal.
Stem cell research can be applied to a number of different areas:
Cancer Research
Sex
determining region Y-box 2 (SOX2) is another important transcription
factor that is critical to the formation of many different tissues and
organs during embryonic development. Numerous studies have been carried
out examining the importance of the SOX2 gene and its relationship to
various cancers such as lung, breast, oesophageal and brain tumours.
Belgian
scientists found that in mice, SOX2 was the most upregulated
transcription factor expressed in stem cells in skin squamous-cell
carcinoma (SCC) or tumours (2). It plays a key role in the initiation
and progression of skin cancer; in normal skin, SOX2 is not expressed,
but begins expression in early stages of tumour formation. When SOX2 is
deleted in mice upon inducing carcinogenesis, a decrease in tumour
formation is noted. This mechanism is different from other SCC due to
genetic alterations.
Non-small cell lung cancer (NSCLC) is a
result of genetic alterations containing stem-like cells that are
responsible for lung tumour initiation, maintenance, relapse and
metastasis (3). In SCC, a subtype of NSCLC, investigators have found
that oncogene, protein kinase C or PRKC1 located at chromosome 3q26 and
SOX2 are coamplified and coordinately over-expressed in lung squamous
cell carcinoma (LSCC) (3). PRKC1 is required for the growth of lung
cancer cells, while tumour protein kinase C iota (PKCι) drives lung
squamous carcinoma cell invasion and transformed growth in vitro and in
vivo. PKCι expression is predictive of poor clinical outcome.
Looking into SOX2
Researchers
found that PKCι phosphorylation of SOX2 led to immediate upregulation
of hedgehog acyltransferase (HHAT). This is required as the
rate-limiting step for activating the signalling pathway. They found
that SOX2-mediated HHAT upregulation drives the hedgehog pathway
activation towards the maintenance of stem-like cells in lung SCC. Three
conclusions resulted from their study:
- PKCι and SOX2 are genetically linked, being co-amplified on chromosome 3q26 in lung SCC
- PKCι and SOX2 are biochemically linked, as SOX2 serves as a substrate for PKC phosphorylation
- PKCι
and SOX2 are functionally linked, as they maintain the stem-like
properties of these lung cancer cells to drive tumourigenesis (4)
The major gene associated with skin melanoma is CDKN2A/p16, cyclin
dependent kinase inhibitor 2A, which is located on chromosome 9p21 (5).
Hedgehog-GLI (HH-GLI) signalling is required for melanoma growth, and
survival and expansion of melanomainitiating cells. It also regulates
genes expressed in embryonic stem cells – including SOX2 – with
transcription factors GLI1 and GLI2. While SOX2 is over-expressed in
melanoma stem cells and knockdown, silencing and depletion of SOX2 leads
to a decrease in self-renewal in melanoma spheres, inhibits cell
growth, induces apoptosis in melanoma cells and significantly decreases
tumour-initiating capability of high aldehyde dehydrogenase activity.
This suggests that SOX2 is required for tumour initiation and for
continuous tumour growth (6).
SOX2 was over-expressed in three
examples of skin SCC, melanoma and LSCC, and played a critical role in
self-renewal, initiation and progression of cancer growth. The absence
of SOX2 demonstrated that cancer cell initiation and growth can be
decreased and perhaps eliminated; however, deleting or silencing the
gene is not feasible, because it is also important for normal cell
growth. Finding the SOX2 on-off switch is crucial to prevent cancer, but
that is easier said than done.
Major Breakthroughs
Since
the completion of the first human genome sequence in 2003, scientists
have been trying to figure out which regions of the genome play an
important part in the development of diseases. They have hypothesised
that the regions that turn genes on and off are key.
Recently,
researchers from the University of Toronto, Canada, discovered the
relationship between the SOX2 gene – critical for early development –
and a region located somewhere else on the genome that effectively
regulates its activity (7). Professor Jennifer Mitchell, lead
investigator, studied how the SOX2 gene is turned on in mice by finding
the region of the genome that is needed to turn on the gene in embryonic
cells.
“Like the gene itself, this region of the genome enables
these stem cells to maintain their ability to become any type of cell, a
property known as pluripotency. We named the region of the genome that
we discovered the SOX2 control region, or SCR,” said Mitchell. “The
parts of the human genome linked to complex diseases, such as heart
disease, cancer and neurological disorders, can often be far away from
the genes they regulate, so it can be difficult to figure out which gene
is being affected and ultimately causing the disease.”
“We then
focused on the region we have since named the SCR, as my work had shown
that it can contact the SOX2 gene from its location 100,000 base pairs
away,” said Harry Zhou, lead author and former graduate student in
Mitchell’s laboratory. “To contact the gene, the DNA makes a loop
(chromatin loops) that brings the SCR close to the gene itself only in
embryonic stem cells. Once we had a good idea that this region could be
acting on the SOX2 gene, we removed the region from the genome and
monitored the effect on SOX2” (7).
Positive Outcomes
Results
revealed that two gene-proximal enhancers – SOX2 regulatory region 1
(SRR1) and regulatory region 2 (SRR2) – showed activity, but SRR2 was
more crucial, as deleting SRR1 had no effect on pluripotency. The assay
also discovered three novel enhancers – SRR18, SRR107 and SRR111 – that
form chromatin loops by forming a chromatin complex with the SOX2
promoter in embryonic stem cells. Only the distal cluster containing
SRR107 and SRR111, located greater than 100kb downstream from SOX2, is
required for cis-regulation of SOX2 in embryonic stem cells (8).
“Just
as deletion of the SOX2 gene causes the very early embryo to die, it is
likely that an abnormality in the regulatory region would also cause
early embryonic death before any of the organs have even formed,” said
Mitchell. “It is possible that the formation of the loop needed to make
contact with the SOX2 gene is an important final step in the process by
which researchers practising regenerative medicine can generate
pluripotent cells from adult cells” (9).
Drug Testing and Development
Human
cells or tissues are used in standard new drugs testing, and cancer
cell lines have been utilised to test anti-tumour medications. However,
the standard method is not always reliable, because there can be varied
responses due to genetic variation from donor to donor, and human cells
are in short supply.
About two years ago, pharma companies
started incorporating human stem and differentiated cells from induced
pluripotent stem cells (iPSCs) when testing new drugs. Researchers from
Edinburgh, UK, were able to generate liver cells that were of the same
quality as cells from human liver tissue to assess drug safety. They
developed a technique to overcome the genetic variability in donor
tissue and were able to make uniform liver cells in the lab (10).
In
addition, GE Healthcare and Stephen Munger, a stem cell biologist,
demonstrated that their heart cells were able to identify which
compounds were toxic out of a set of compounds in a blind trial (11).
Therefore, using iPSCs gives researchers the ability to perform drug
testing on a wide range of cell types.
Organ-On-Chip
Currently,
scientists at the Wyss Institute are developing miniature organs using
stem cells called ‘organ-on-chip’ for drug testing and development (12).
The goal behind this is to eliminate animal model testing since it is
not representative of the human system, and is expensive and
time-consuming to perform. Eventually, they hope to develop ten organs
on a chip, and link them together to mimic the human body in safety and
efficacy testing.
Unfortunately, the creation and scaleup of
quality stem cells is difficult to achieve. To screen for drugs
effectively, scientists must be able to create identical conditions by
precisely controlling cell differentiation. However, the signalling
mechanism for controlling differentiation is not well-known in some cell
types and tissues, making it harder to mimic identical conditions. More
work needs to be done before researchers can achieve this.
For Transplantation
Investigators
are trying to grow adult stem cells in cell culture and manipulate them
to generate specific cell types to treat injuries and diseases.
Potential approaches include regenerating bone using cells derived from
bone marrow stroma, developing insulin-producing cells for type 1
diabetes, and repairing damaged heart muscle following a heart attack
with regenerated cardiac muscle cells.
However, in each tissue,
there are a very limited number of stem cells, and once removed from the
body their capacity to divide is limited, thus making it difficult to
generate large quantities. If scientists can reliably direct embryonic
stem cells to differentiate into specific cell types, they may be able
to use these to target certain diseases in the future. Conditions that
could be treated include diabetes, traumatic spinal cord injury,
Duchenne muscular dystrophy, heart disease, and vision and hearing loss.
If scientists can use the patient’s own embryonic cells or
iPSCs, and differentiate them into specific cell types before
reintroducing them into the body – much like immunotherapy – there would
be a lower chance of rejection by the immune system. This would be a
significant advantage, as the only recourse to rejection is the
continuous administration of immunosuppressive drugs, which can have
deleterious side-effects.
Promising Future
In
2006, Shinya Yamanaka and colleagues made a breakthrough discovery by
showing that skin fibroblasts can be reprogrammed into embryonic stem
cell-like cells. These induced iPSCs hold tremendous promise for
regenerative medicine and drug discovery. However, being able to control
cell proliferation and differentiation predictably and safely, will
require more basic research into the molecular and genetic signals that
regulate cell division and specialisation.
Transcription factors
– such as Oct4, Nanog, SOX2, KLF4 and c-Myc – are crucial for the
regulation of embryonic development and cell differentiation. These same
factors also play important roles in cancer biology. As noted above,
SOX2 over-expression is involved in selfrenewal, initiation and cancer
progression, and manipulation of SOX2 significantly influences the fate
of cancer cells.
It has also been demonstrated that the SOX2
regulatory region – particularly the SRR2 – determines the fate of
cells, as deletion of SRR1 does not affect pluripotency. An abnormality
in the regulatory region not only leads to embryonic death, but may
contribute to the initiation of cancer cells and the binding of the
chromatin loop that starts and continues the process of development.
Finding methodologies to turn off this cis-regulation at the appropriate
time and duration – without affecting normal cells – could be an interesting next challenge.
Techniques
used to develop iPSCs have given us a greater understanding of how to
control some aspects of cell proliferation and differentiation, but
further research is also needed in order to be able to manipulate cells
predictably and safely. Unanticipated mishaps may lead to cells of the
wrong type that may cause rejection in cell transplantation or, even
worse, cancer.
Currently, viruses are used to introduce the
reprogramming factors in adult cells. However, in animal studies, the
virus used to introduce stem cell factors sometimes causes cancers, and
researchers are currently investigating non-viral delivery strategies
(1).
We have observed that SOX2 is genetically, biochemically
and functionally linked to PKCι in driving tumorigenesis in the LSCC
study. This complexity reinforces the need to fully understand the
mechanism and process of cell proliferation and differentiation. If
scientists can control the pertinent transcription factors’ on-off
switch like SOX2, it will not only reveal the pathway to potential
cancer therapies, but also the ability to consistently create uniform
differentiated cells. This is critical in testing new drugs for safety
and efficacy, and ensuring that cells for transplantation are effective
and safe.
References
1. National Institute of Health, Stem cell information. Visit: http://stemcells.nih. gov/info/basics/pages/basics1.aspx
2. Boumahdi S, SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma, Nature 511: pp246-250, 2014. Visit: www.nature.com/ nature/journal/v511/n7508/full/ nature13305.html
3. Justilien V et al, The PRKCI and SOX2 oncogenes are coamplified and cooperate to activate hedgehog signaling in lung squamous cell carcinoma, Cancer Cell 25(2): pp139-151, 2014. Visit: http://ac.elscdn.
com/s1535610814000336/1-s2.0- S1535610814000336-main.pdf?_
tid=a2ce8be8-fc00-11e4-ba77-00000aab0f27&acdnat=1431804218_16492a67938967
f4c38000318e8fdbb8
4. Fields A and Justilien V, Interview on
oncogenes driving squamous cell lung carcinoma, Cancernetwork. Visit:
www. cancernetwork.com/lung-cancer/ oncogenes-driving-squamous-celllung-
carcinoma
5. National Cancer Institute, Genetics of skin cancer – For health professionals (PDQ®). Visit: www.cancer.gov/types/ skin/hp/skin-genetics-pdq#link/ _41_toc
6. Santini R et al, SOX2 regulates selfrenewal and tumourigenicity of human melanoma-initiating cells, Oncogene 33(38): pp4,697-4,708, 2014. Visit: www.ncbi.nlm.nih.gov/pmc/articles/ pmc4180644
7. University of Toronto, Cell biologists discover on-off switch for key stem cell gene, ScienceDaily, 15 December 2014. Visit: www.sciencedaily.com/ releases/2014/12/141215084946.htm
8. Zhou HY et al, A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential, Genes & Development 28(24): p2,699, 2014. Visit: http://genesdev.cshlp.org/ content/28/24/2699
9. On-off switch for critical stem cell gene discovered, Genetic Engineering & Biotechnology News,
15 December 2014. Visit: http://genengnews.com/
gen-news-highlights/on-offswitch- for-critical-stem-cellgene-
discovered/81250699
10. University of Edinburgh, Stem cells reach standard for use in drug development, ScienceDaily, 11 June 2013. Visit: www.sciencedaily.com/ releases/2013/06/130611111712.htm
11. Cressey D, Stem cells take root in drug development, Nature, 2012. Visit: www. nature.com/news/stem-cells-take-rootin-drug-development-1.10713
12. Wyss Institute, Organs-on-chip, 2015. Visit: http://wyss.harvard.edu/ viewpage/461
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