Stem Cells: Flexible friends
Stem cells are powerful tools in biology and medicine. What can scientists do with these cells to exploit their incredible potential?
Adapted from Principles of Biology published by Nature Education.
At
first, people and objects seem fuzzy around the edges. Faces become
unrecognizable in low light. Reading and driving are no longer possible.
Eventually, darkness descends.
A disease called macular degeneration is responsible for this progressive loss of vision. The light-sensitive cells in the eye, located in a thin layer of tissue called the retina, are damaged and stop working. Macular degeneration is mainly a disease of ageing and is the leading cause of vision loss in people aged 65 and older. It is expected to affect nearly 3 million people in the United States by 2020.
Related diseases, including a genetic condition called Stargardt's
macular dystrophy, affect young people as well. With this condition,
fatty deposits build up behind the retina, causing it to degenerate, and
vision loss is inevitable. What if those dying cells in the retina
could be repaired or replaced? Scientists are testing a daring new
treatment for both macular degeneration and macular dystrophy. They are
injecting replacement cells into the back of the eye to repair the
retina. The company running the trials, Advanced Cell Technology, makes
the replacement eye cells from human embryonic stem cells.
Stem
cells are powerful. They are pluripotent, which means they can transform
into any one of the 220 cell types in the human body. One stem cell can
also divide to produce millions more stem cells. The potential for stem
cells to renew themselves, or to create new tissue, is almost infinite.
These properties make stem cells an important tool in the lab and in
medicine. They offer scientists a better understanding of human
development and a way to test drugs without putting human volunteers at
risk, and provide a way of replacing damaged tissues, such as the cells
of the retina, muscle or spinal cord. But how can we make the best use
of stem cells? How can we make them grow where we want, grow how we
want, and repair damaged or diseased tissue?
To understand stem
cells, we first must establish their basic biology. There are stem cells
in nearly all animals, from tiny worms to mice and humans. There are
three types of stem cell — embryonic stem cells, adult stem cells, and
induced pluripotent stem (iPS) cells — each with its own capabilities
and limitations. An embryonic stem cell comes from an organism at its
earliest stages of development. When a sperm meets an egg and the
resulting zygote begins to divide, each cell holds the potential to
become any cell type in the body. This is the essence of pluripotency.
Not until the cells divide a few times do they start to lean towards one
fate or another, expressing genes specific to one cell type. This
process is called differentiation.
Once an embryo has developed
into a mature organism, most cells have undergone differentiation, but
some cells are special. Unlike most adult cells, they retain the ability
to multiply and become other types of cell. These adult stem cells
reside in special stem-cell niches, regions of certain tissues where
they wait for cues from the organism to replace or repair tissue. They
are usually found in tissues that must continuously replenish
themselves, such as the blood, the skin and the gut. But they have also
been found in the brain, which replaces its cells much less frequently.
These adult stem cells are said to be multipotent — unlike pluripotent
cells, they cannot turn into any one of the 220 cell types in the human
body. Neural stem cells in the brain can differentiate into several
kinds of brain cell but could not become liver cells, for example.
In
the past decade, scientists have also learned to make stem cells from
regular mature, differentiated cells. These cells are called induced
pluripotent stem cells because scientists force them to become
pluripotent even after they have reached a differentiated state. By
turning up the expression of just a few genes, scientists can force a
skin cell, for example, to retrace its developmental pathway backwards
all the way to a flexible pluripotent state.
The ability to
manipulate the fate of a cell has led to much excitement about the
potential of these fascinating cells. But stem-cell research is an
emerging field, with many fundamental questions still unanswered. For
example, how do the three types of stem cells differ, and how are they
the same? Can we use these cells to cure diseases? And can we use them
to rebuild tissues or organs?
An eye on development
Every
cell in the body has the same set of genes. An eye cell is different
from a liver cell because they differ in which genes are turned on and
which genes are turned off. In a retinal cell, genes that enable light
sensing are turned on, and genes that make digestive proteins are turned
off. In liver cells, the opposite is true. The scientists who are using
embryonic stem cells to cure blindness have figured out which genes are
on and which genes are off in the retinal cells that patients need. By
growing pluripotent embryonic stem cells with chemicals and proteins
that make them differentiate into retinal cells, the researchers have an
unlimited supply.
The ability to do this is the result of decades
of research, going back to the discovery of stem cells. In 1868, German
biologist Ernst Haeckel first used the term 'stem cell' to describe a
zygote. Researchers have been working with mouse embryonic stem cells
since 1981 but the field took off in 1998, when researchers isolated
human embryonic stem cells for the first time1.
The
breakthrough came when James Thomson and his team at the University of
Wisconsin–Madison obtained a group of cells called an 'inner cell mass'
from a blastocyst, which is a natural source of stem cells. A blastocyst
is a very early stage embryo, created here from the controlled
fertilization of an egg by a sperm in the laboratory. It is simply a
hollow ball of cells with a cluster of cells inside. Scientists at
fertility clinics often make more embryos than prospective parents will
need to get pregnant, and the surplus embryos are usually frozen or
discarded, according to the parents' wishes. In this case, the parents
offered their surplus blastocysts to science. If the blastocysts were
implanted into a uterus, the inner cell mass would become a fetus and
the outer shell, or trophectoderm, would form accessory tissues such as
the placenta. Without implantation, however, the blastocysts can be
converted to cell cultures.
To do this, the researchers removed
the inner cell mass and grew the cells in a flat dish create a cell
line, a set of genetically identical cells. This required a
nutrient-rich liquid and some mouse cells to serve as feeder or support
cells.
The isolated cells grew and grew. Indeed, part of the
definition of a stem cell is that it can divide almost infinitely,
producing more and more stem cells. In contrast, ordinary cells die
after a certain number of divisions, limited by telomeres, or caps on
the DNA. The cell's DNA replication machinery cannot reach the very tips
of the strand, so the telomeres shorten a bit with every cell division.
Stem cells, however, have an enzyme called telomerase that builds the
telomeres back up, essentially making the cells immortal.
But
there is more to being a stem cell than being able to divide forever;
the cells must also be pluripotent, having the capacity to become any
type of cell. During embryonic development, all the body's organs and
tissues develop from one of the three germ layers: endoderm, mesoderm
and ectoderm. A pluripotent cell can form all three germ layers. A cell
that can develop only one germ layer has already started down the road
toward differentiation and is not pluripotent.
Scientists often
test for pluripotency by injecting the cells into a mouse. Once in the
mouse, pluripotent cells will form a teratoma, a clump of cells
containing all three germ layers. Thomson's cells passed the test. The
researchers had discovered how to produce embryonic stem cells.
Scientists
now have a supply of embryonic stem cells that will divide indefinitely
and are pluripotent. They know how to isolate them and work with them
in the lab, and they know how to keep them healthy in the correct
culture conditions. So why haven't they already turned embryonic stem
cells into every type of cell they need to cure disease?
Unfortunately,
it's not that easy. In the case of the retina cells needed to cure
macular degeneration, researchers have discovered which molecules can
coax stem cells to become the correct cell type for transplantation into
the eye. However, they have not yet discovered the specific conditions
and transplant techniques for all 220 of the cell types in our bodies.
Different cell types require different conditions and molecular cues. As
well as the technical limitations regarding the cell culture,
scientists are held back by the human culture outside the lab because
not everyone supports research on embryonic stem cells.
Objections to stem cell reasearch
What
concerns do people have about the use of embryonic stem cells? The main
objections are based on people's cultural and religious beliefs.
Catholics
and some conservative Protestants, for example, oppose stem-cell
research because they believe that human life starts at conception,
making it unethical to destroy a zygote or an embryo, or even a
blastocyst. People from other religions, including Judaism, Islam and
Hinduism, view the embryo differently and are generally not opposed to
stem-cell research. Still others object to the research because they are
misinformed about the source of stem cells, believing they come from
aborted human fetuses rather than blastocysts that have never been
implanted. In addition, many scientists working on stem-cell research
also hold strong religious beliefs, so it is not as simple as science on
one side and religious beliefs on the other.
The research
guidelines for stem-cell research in the United States are set by the US
government, which funds most of the country's medical research. Not
surprisingly, its view on embryonic stem-cell research alters with
changes in presidential administrations. In 2001, President George W.
Bush authorized the federal support of human embryonic stem-cell
research only if the cells were derived before 9 August 2001 from an
embryo that was created for reproductive purposes and was no longer
needed. In addition, people donating embryos for research had to give
informed consent by signing a document saying they know what the embryos
would be used for, and they could not receive any money in return.
These
restrictions limited the availability of human embryonic stem-cell
lines to fewer than a hundred. In 2009, President Barack Obama issued an
executive order so the federal government could support and conduct
responsible, scientifically worthy human stem-cell research, including
embryonic stem-cell research, to the extent permitted by law. Even so,
fewer than 200 cell lines are available for federally funded research.
Scientists who need cell types not included in the federal registry must
seek funding from states or private foundations. The Stanford
Encyclopedia of Philosophy has published a thorough review of the
ethical questions raised by stem-cell research, and the website of the
US National Institutes of Health (http://stemcells.nih.gov/info/ethics.asp) offers several resources on the topic.
For a long time, scientists thought that once a cell differentiated, it could never regain its pluripotency. They assumed that it must be set in its identity because it had so many genes turned on and off in ways particular to that cell type. In 2006, however, Kazutoshi Takahashi and Shinya Yamanaka of Kyoto University in Japan proved otherwise. They turned mouse skin cells — specifically, fibroblasts — into stem cells. These were the first induced pluripotent stem (iPS) cells.
How do we rewrite a cell's developmental programming? The researchers
thought that if they could force a cell to turn on the genes that are
turned on in stem cells, and turn off all the other genes, that cell
would become a stem cell. So they needed to know which genes are crucial
to the stem-cell phenotype. What genes make a stem cell a stem cell?
The
scientists selected 24 genes that they thought might contribute to a
stem cell's pluripotent nature. They turned on these genes in the mouse
fibroblasts, in many different combinations. In the end, they found that
a mix of four genes that encode transcription factors — molecules that
turn genes on and off — could turn fibroblasts into iPS cells. The genes
were Oct3/4, Sox2, c-Myc and Klf4.
Yamanaka
and colleagues still needed to prove that their iPS cells were fully
pluripotent, and that meant showing they could form an entire animal the
way embryonic stem cells do. They added some of their iPS cells to a
developing mouse embryo and showed that the iPS-derived cells formed
part of the resulting mouse. Two other teams published similar results
in the same week.
The iPS cell research race was on, and although
Yamanaka had started it, his group soon had to keep pace with a crowd of
other scientists excited by his discovery. Several investigators are
now reprogramming cells to make their own iPS cells. Harvard University
and other universities have entire centres devoted to iPS studies. One
of the great benefits of iPS cells is that they do not need donated
embryos.
Scientific progress often accelerates when several groups
are exploring the same question. The stress to make an important
discovery and publish first can be overwhelming, but that kind of
motivation can spur a field on. Different research groups can confirm
one another's results, build a case for well-supported theories, and
quickly expose incorrect hypotheses.
The next immediate question
was whether iPS cells can be made from human tissue. In 2007, three
papers published in rapid succession reported the making of human iPS
cells. Yamanaka and one other group used the original recipe2, whereas a third research team, led by Thomson, used a different technique.
Progress
was rapid. It took less than six months for scientists to modify the
mouse iPS protocol to work for human cells. In contrast, it had taken 17
years to move from the first mouse embryonic stem cell to the first
human embryonic stem cell, obtained by Thomson in 1998.
Now that
they knew how to make human iPS cells, researchers wanted to improve the
process. Some parts of the methods might be dangerous to patients who
might one day be candidates for stem-cell therapies. The two main
concerns were genes that can cause cancer and the use of viruses to
deliver these genes via viral vectors. One of the genes that Yamanaka
and others used to make iPS cells was c-Myc, a cancer-causing oncogene.
Mice made from iPS cells that have high levels of the c-Myc protein
frequently develop tumours, probably because c-Myc encourages not only
stem-cell production, but also cancerous growth. Because of the
similarities between stem cells and cancer cells, the possibility that
stem cells could turn cancerous is a major concern for scientists.
In
2008, Yamanaka and his colleagues announced that they had managed to
eliminate c-Myc from their recipe, rather by accident. In a series of
experiments, they discovered that Myc is not essential but merely speeds
up the process, so their technique could work without it.
Some of
the early iPS methods, including one developed by Thomson and his team,
relied on viruses to carry the newly created DNA into the human cells.
Could these viruses have a negative effect effect by activating cancer
genes or causing some other undesirable gene expression? Some research
groups have sidestepped this concern and developed virus-free
techniques. For example, in 2008, the Kyoto researchers showed that they
could move stem-cell genes into human genes without viruses by using
plasmids, circular DNA molecules that can hop between cells and into
genes. Like viruses, however, plasmids might also enter the genome at
the wrong place, accidentally activating cancer genes. To avoid this
risk, researchers later created yet another method for delivering
stem-cell genes. They first inserted stem-cell genes into cells using
non-integrating vectors or plasmids that can be cleaned out or removed
once the cells had become pluripotent. This change eliminated the risk
imposed by inserting a nucleic acid or plasmid3.
It
would be better still to get rid of the gene-insertion technique
altogether and rely on chemical factors in the growth medium to force
cells towards the pluripotent state by changes in the transcription of
their own genome. So far, researchers working with mice have managed to
replace the signals normally activated by transcription factors with
chemical signals. Amazingly, they have replaced everything except the
effects of transcription factor Oct3/4.
Cell memory
Just
like embryonic stem cells, iPS cells can divide indefinitely and
differentiate into the three germ layers. Unlike embryonic stem cells,
however, iPS cells used to be differentiated cells. Might they retain
some genetic or cellular 'memory' of their previous life as a skin cell
or a muscle cell? Recent studies suggest that they do, in the form of
epigenetic markers on the DNA. These markers are methyl, acetyl and
other chemical groups that attach to the DNA, turning some genes off and
others on. Cells acquire epigenetic markers as they differentiate, and
they maintain some of them when they dedifferentiate into iPS cells.
The
discovery that iPS cells retain some memory was serendipitous. George
Daley of Harvard University noticed that he had more success making
blood cells from iPS cells that used to be blood cells than with iPS
cells that started out as skin cells. Daley's team and other groups
found that iPS cells keep some of the epigenetic markers that turned
genes on or off in the original, differentiated cells. How can we
control these markers? Only when the markers are gone will scientists
truly be able to say that iPS cells are no different to embryonic stem
cells.
Many of the intended uses of stem cells involve
transplanting them into patients. But concerns about the risk from
cancer-causing genes leave some scientists wondering if they should
bother with the iPS stage at all. If doctors could avoid a pluripotent
step in their treatment, it would be less risky. Ideally, they would
like to avoid removing and transplanting cells at all. Is it possible to
use drugs to trigger someone's own cells to redifferentiate into the
cell type they need?
This idea is starting to look plausible.
Scientists have recently shown that it is possible to skip directly from
one differentiated cell type to another. This approach, called
transdifferentiation, is very appealing, and many scientists are working
on it.
In 2008, Qiao (Joe) Zhou and Douglas Melton at Harvard
University managed to turn one type of pancreatic cell in mice into
another, the beta-islet cells of the pancreas that produce insulin.
These are the cells that are destroyed in people with type 1 diabetes.
Performing the process inside the animal is advantageous because the new
cells can develop in their natural environment instead of in a lab
dish.
Despite its medical significance, changing one pancreatic
cell type to another is a fairly small jump between similar cell fates.
Researchers led by Marius Wernig at Stanford University made a bigger
leap in 2010 with a protocol to turn mouse fibroblasts into neurons. A
year later, they accomplished the same feat with human cells. The
neurons they made, however, are not exactly the same as any particular
kind of brain cell; instead, they had a mixture of neural
characteristics. In the same year, another group of scientists managed
to transdifferentiate fibroblasts into the specific type of neuron that
produces the neurotransmitter dopamine. Dopamine's functions include
controlling movements, and these neurons are lost in people who have
Parkinson's disease.
Researchers are still not sure whether the
cells change directly from one type to the next, or whether they go
through other, less-differentiated stages. And there is still no
tried-and-true, reproducible method. More work is needed to make these
cells useful for detailed studies or medical use.
Adult stem cells
Researchers
are also trying to take advantage of the adult stem cells we all have
in our bodies. These adult stem cells do not raise the ethical questions
that embryonic stem cells do because no embryos are needed. The problem
is that adult stem cells exist in very small numbers and are often
buried deep in the tissue. Progress in this area has been hindered by
lack of sufficient cells, but doctors have been using adult stem cells
to help patients for about 40 years. They are mainly used for two
treatments: bone-marrow transplants to rebuild a patient's immune
system, and skin grafts to replace skin over a burn or other injury.
Scientists
would like to develop other applications for adult stem cells. However,
they still do not fully understand where adult stem cells come from and
how they differentiate when needed. If we can solve these mysteries,
doctors might be able to use drugs to activate a patient's own adult
stem cells to perform the necessary repairs.
For example, a heart
attack damages the heart muscle cells. Ideally, adult stem cells in the
heart should be able to rebuild the muscle. For now, though, adult stem
cells are limited to replacing a small number of nearby cells, not the
vast damage done by a heart attack. In 2011, scientists discovered a
protein that activates those adult stem cells to make new heart cells.
If they can do this on a larger scale, doctors might be able to make the
heart repair itself.
Medical applications
Stem cells can
teach us about the biology of pluripotent cells, how the human body
develops, and what happens during disease. They are also a tool for
developing safer ways to design and test new medications. Finally, they
are the basis for regenerative medicine, using stem cells to repair
damaged tissue.
They have enabled scientists to work out how to make their favourite differentiated cell types. In my laboratory, we study the cells that form bone and cartilage. To make these cells, we first allow colonies of stem cells — either embryonic or induced pluripotent stem cells — to grow in free-floating suspension cultures (as opposed to flat on a dish). This treatment causes them to start differentiating into cells of the three germ layers. Then we use growth factors to turn the cells into mesenchymal cells, an intermediate stage in the transformation to bone, cartilage and fat. Finally, we add chemicals that encourage the differentiation of bone or cartilage cells. The next step is to find out whether these cells will make bone and cartilage in an animal instead of a lab dish. We are injecting the not-quite-differentiated cells into mice to see if they continue to differentiate and form the correct structures. Other labs are exploring how stem cells can be used to repair bone and cartilage damage and heal joint and bone diseases.
Over the past decade, scientists have developed recipes to guide stem
cells towards one fate or another, including cells of the heart, liver,
brain and pancreas. They can then study both healthy and diseased
versions of those cells. It is often easier to study individual cells
than an entire person or animal. If scientists have a sample of skin
from a patient they can turn it first into iPS cells and then into a
cell type relevant to disease.
For example, scientists interested
in spinal muscular atrophy want to study the motor neurons affected by
this condition, which causes patients to lose some lower motor neurons,
resulting in muscle weakness, paralysis and often death. One group, led
by Allison Ebert of the University of Wisconsin–Madison, derived iPS
cells and then created motor neurons from both a patient with the
disease and his mother, who had no disease. The motor neurons grown from
the patient's cells maintained the genetic characteristics of the
disease and showed selective deficits compared with those derived from
his mother4. Similar studies have been conducted in patients with another nervous system disorder known as familial dysautonomia.
These
disease-specific cells are an important tool for drug discovery.
Researchers can screen hundreds of drugs on iPS-derived motor neurons
and pick the ones that seem to alleviate the pathology in the diseased
cells. This kind of experiment would be unethical in humans and
prohibitively expensive in animals, but it is relatively easy with
cultured cells.
Researchers at Harvard University's Stem Cell
Institute have made cell lines representing more than ten diseases,
including type 1 diabetes, Down's syndrome and muscular dystrophy, and
they plan to make many more to share with other stem-cell researchers.
Another cell repository, supported by the National Institutes of Health
and housed at the Coriell Institute for Medical Research in Camden, New
Jersey, is also sharing iPS cells that represent diseases, including
Huntington's disease and spinal muscular atrophy.
Stem cells also
offer an opportunity to screen drugs for side effects before they are
tested in people. For example, researchers can turn stem cells into
heart cells that are differentiated enough to pulse rhythmically like
the heart, even when growing in a dish. If a drug damages these cells,
it is cause for concern and the drug will probably not move forward to
testing in patients. Pharmaceutical companies see stem-cell-derived
cultures as a way to streamline the drug testing process and make it
safer. The field of drug discovery and disease models using iPS cells is
moving quickly and is way ahead of regenerative medicine.
Towards a treatment
Doctors would like to use stem cells to treat conditions such as
macular degeneration and spinal cord injuries. The few clinical trials
already underway represent only a fraction of what will one day be
possible using stem cells. For now, the goal is to ensure that stem-cell
treatments are safe, making sure that the cells won't end up in the
wrong place, cause an immune reaction or develop into cancer.
Regarding
stem cell-based treatments for macular degeneration, Advanced Cell
Technology is taking advantage of the eyes being a relatively safe place
to start. Stem cells are less likely to be rejected in the eyes than in
other organs because they are separated from the immune system by the
blood–brain barrier. In addition, doctors already have all the tools
they need to look into the eye and make sure the transplanted cells are
behaving properly.
However, there are several steps between thinking of fixing blindness
with stem cells and ultimately using them as a cure in patients.
Scientists began by designing a recipe to make retinal cells from
embryonic stem cells. They then tested the treatment in animals. Some
rats have a version of macular degeneration, and some mice have a
condition that is similar to Stargardt's macular dystrophy. When the
scientists injected their retinal cells into these animals' eyes, the
cells gathered near the retina and improved the animals' sight. Now the
scientists are testing the safety of the treatment in a small number of
people. If they can prove that the cells are safe, they will test them
in more patients and find out if they actually improve vision.
In
another exciting avenue of research, a company called Geron is running a
clinical trial using nerve cells derived from embryonic stem cells to
treat spinal cord injuries. The company is collaborating with
researchers at the University of California, Irvine, led by Hans
Keirstead. Nerves rarely grow back after injury because they lose their
protective coating of myelin protein, which insulates nerves, much like
the rubber insulation on an electrical wire.
Cells called
oligodendrocytes are responsible for making myelin and protecting nerves
from infection. The scientists reasoned that if they could replace
these supportive cells they could restore the myelin around the nerves
and release growth factors to repair them. First, of course, they had to
work out the recipe to coax an embryonic stem cell into becoming an
oligodendrocyte. After much trial and error, Keirstead's team succeeded.
When they transplanted the freshly made oligodendrocytes into rats
after a spinal cord injury, new myelin formed around the injured nerves
and the rats regained some movement. Geron is now testing the safety of
the cells in patients.
To reduce the likelihood of rejection
during tissue transplant, researchers are considering using iPS cells as
patient-specific transplantable material. Scientists could
theoretically correct the genetic defect in iPS cells from a patient and
then transplant them back into the same person. For example, Rudolph
Jaenisch's research team at the Whitehead Institute for Biomedical
Research in Cambridge, Massachusetts, corrected the genes of mouse iPS
cells to treat mice with sickle-cell anaemia. The work provided proof of
principle that someday iPS cells from someone with a disease could be
corrected and then transplanted back into the same person.
The tip of the iceberg
The
discoveries and vast implications of the research described above are
only the beginning. Innovative researchers around the world are
exploring the impressive capabilities of stem cells by asking a range of
questions.
First, what happens to transplanted stem cells? Do
they go to the correct place and perform the correct functions? Second,
can we ensure that stem-cell treatments do not cause cancer or other
ailments? Researchers are making progress in their efforts to refine
stem-cell recipes to reduce the need for cancer-causing oncogenes or
viruses. Ensuring safety is essential before stem cells are used in
large numbers of patients. Third, can iPS cells be used instead of
embryonic stem cells, or do they retain too much 'memory' of their
former lives? Fourth, will the immune system reject stem-cell
transplants? And what are adult stem cells, and where do they come from?
How can we get a better supply of them, and can we use drugs to make
them work better?
These questions are only a small subset of those being explored in research laboratories. And there are bound to be exciting questions to pursue that we just haven't thought of yet. This growing field of research with a variety of applications in medicine has captured the attention of our culture with its potential to offer hope to those with incurable injury and disease. Clearly, stem-cell research has changed our perspective on what is possible.
