Stem Cell Research

Sorry for the long absences, but my priorities lie elsewhere, especially since I’m not actively teaching these days. I will still post stuff, but it will be at a relaxed pace since I only have the time to write during random bouts of insomnia.

Anyway, the topic here is stem cells, since it was requested sometime ago. I will break my usual rules and actually pander to my audience by focusing more on implications for humans and medicine. There is little developmental biology and I will not talk about organo- or morphogenesis (as I would normally do when talking about stem cells).

Before we start, two disclaimers!

Disclaimer 1: This is an introduction, not a detailed analysis and review of all genetic, epigenetic and biochemical pathways we know of. I can go into more detail about them, but only if you ask for it!

Disclaimer 2: I will not be discussing any bioethical problems here. Interesting as they are, I do not feel they belong with a scientific introduction to stem cell research, since it might turn this into a festering cesspool of philosophical wankery.

Stem cell research has come into the spotlight over the past few years because of new discoveries that revolutionised our understanding of how they work: we found out that adult stem cells are not predetermined, as was thought before, but are plastic: instead of being restricted to forming their host tissue’s cell type, they can (under the right influence) differentiate into all cell types – this is where stem cells’ medicinal potential lies.

This may come as a surprise, but there is no set, agreed-on definition for stem cells. This has several reasons, from the history of embryology to the overall diversity of stem cells. I will be using the definition of stem cells that the public understands to avoid confusion and overcomplication: a stem cell can self-renew and it can differentiate into one or more cell types.

At the top of the hierarchy are totipotent cells: this is the fertilised ovum. It can generate the three germ layers (endo-, meso-, ectoderm), germ cells and also the trophoblasts (the cells lining the blastocyst; they provide nutrients to the embryo and later develop into part of the placenta).

The stem cells that cause all the ruckus are the embryonic stem cells (ES). They are pluripotent, meaning they can generate any differentiated adult cell. They can be cultivated and maintained in their undifferentiated state in a cell culture, since there is no limit to how much they can renew themselves.

The standard ESs are derived from the blastocyst – the stage before an embryo is implanted into the uterus wall – since most of the cells there are undifferentiated. There is another, recently discovered source of pluripotent stem cells, namely from the epiblast (epiSC). They can also be taken from primordial germ cells (EG), which are the precursors to the gametes (sperm and egg).

I will not go into embryology and morphogenesis to describe how the adult forms from the zygote. In adults, most of the organs also have stem cells (called stromal stem cells or mesenchymal stem cells (MSC); I will be referring to them as somatic stem cells). However, they are limited in their differentiation range, in that they can only generate mesodermal tissues (e.g. bone). They can be multipotent, like hematopoietic cells (stem cells that can only differentiate into the different types of blood cells) or unipotent, like skin cells (they can only differentiate into more skin cells, which is what happens when you cut yourself).

The other characteristic of stem cells is their ability to self-renew to maintain the stem cell population (or increase it in the embryo). Their differentiation range stays the same after renewal: a unipotent stem cell will not generate a multipotent one. When discussing stem cell renewal, it is important to note the difference between potential and reality: neural stem cell cultures can be grown to enormous sizes, but those renewal rates are never observed in vivo. Of course, making stem cells achieve their full potential under physiological conditions is one of the ultimate goals, but in order to do that, we first need to understand how the renewal process is controlled.

One thing we know is that ES cells have a very fast cell cycle: the G1 (growth) phase is very short, so the S phase is reached quickly. This comes at the cost of safety though, since many checkpoints are skipped. If we want to make MSCs pluripotent, this may be one of the steps required.

Somatic stem cells are different, since they need to actively respond to environmental changes. Going through such a reckless cell cycle would cause cancer, so their renewal is much more regulated. They need to be able to go into resting phases for a stretch of time and be activated when needed (activated means they need to get back to a regular cell cycle). As I mentioned before, somatic stem cells retain their identity. This is also another target of research: it’s known that ES cells actively repress gene expression to stay undifferentiated. If we find out what’s being repressed, we can get another way to manipulate stem cells.

In vivo, some stem cell types are plastic (as I mentioned at the beginning). Stem cells from the bone marrow can differentiate into various mesodermal tissues: bone (cell type: osteoblast), cartilage (cell type: chondrocyte) and fat (cell type: apidocyte), skeletal muscles, heart tissue (cell type: cardiomyocyte) and endothelium (blood vessels). Outside of the mesoderm, there is evidence that bone marrow stem cells can differentiate into neuronal cells (germ layer: neuroectoderm) and to epithelial cells (endoderm).

Other stem cell types show lesser degrees of plasticity, but the fact that they can all be artificially triggered in a culture to go further than their counterparts living in an organism shows that the possibilities are great.

One of the goals of stem cell research is to make adult stem cells pluripotent. Sometimes, it is as simple as putting one cell type in another tissue – as with fusing blood cells with liver cells to repair liver damage – but this is the exception rather than the rule, at least in humans (animals capable of regeneration are extreme cases).

The major advance in this area came from induced pluripotent stem cells (iPS). These are adult stem cells that have been artificially grown to become pluripotent (by changing their gene expression to match that of ES cells). The potential here is amazing and basically gets rid of the only argument moral opponents of stem cell research ever put forward, since  no ES cells will need to be harvested.

As things stand now, we can reprogram cells from most tissues to become pluripotent. IPS cells hold great promise for personalised medicine, since there is no risk of host rejection. Talking about the possibilities iPS cells offer us will take up pages, so I’ll just let the following picture do it.

But with all the excitement involving iPS cells, let’s not forget about other ways to induce pluripotency, outlined in the diagram below.
The basic fact behind all of these strategies is that a differentiated cell’s genome is no different from a stem cell’s genome – so they should both have the same potential. Of course, this doesn’t make the task easy, and all these processes are rather inefficient.

Nuclear transfer refers to removing the nucleus of an egg cell and injecting a somatic cell’s nucleus in its place. This egg cell can then be used to impregante an animal, giving rise to a clone , or it can be grown in culture as a source of embryonic stem cells (theis process is also referred to as SCNT: somatic cell nuclear transfer).

We can make a somatic cell–ES cell hybrid by cell fusion, giving us pluripotent cells. The mechanism behind this is not fully understood, but it does have something to do with the epigenetic state of the ES cell. However, it is generally not preferred due to its inefficiency and the resulting tetraploidy – making them diploid again is too risky (because of genome destabilisation).

Explantation refers to the culturing of multipotent cells (e.g. spermatogonial stem cells). Finally, we can customise viruses to infect somatic cells and induce pluripotency (i.e. iPS cells).

At the end of the day, the way forward is to further our understanding of how our treatments work at the molecular and genetic level (see picture below).

One thing you may notice (and which I touched on already) is that many of these depend on growing cells on petri dishes or in test tubes. Therefore, what we find out using them does not reflect in vivo conditions – this is something that should always be kept in mind, and is one of the reasons why animal and human testing is so important.

For all the controversy surrounding them, stem cell therapies are common. Take bone marrow transplants, for example. They are used to treat leukaemia and bone marrow failure,as well as to help rebuild the immune system in patients undergoing chemotherapy and radiation therapy. A bone marrow transplant is nothing more than the injection of multipotent hematopoietic cells from the bone marrow or blood.

That brings us to the research questions behind stem cell research. Before getting to any medical application, the biological underpinnings of any treatment have to be very clearly understood.

One aspect is the genetic regulation of stem cells: what is it about the gene expression in a pluripotent cell that gives it the flexibility to differentiate into all the other cell types? What genetic pathways underlie stem cell regeneration? Pieces of the answers are pouring in every day thanks to efficient DNA sequencing technology and the associated growth of bioinformatics, and we now know about the genetic core controlling pluripotency (see figure below).

We also need to figure out the epigenetic states of ES cells. As a reminder, epigenetics is the heritable modification of DNA that doesn’t affect the DNA’s sequence. By comparing ES and MSC cells, we should be able to pinpoint where the differences lie.

This research can then be used to figure out the mechanism behind iPS cells. Some cell types are more readily induced to become pluripotent. It could be due to their gene expression patterns, which are controlled by transcription factors. One important element of this is determining how big the role of genetics is and how big the role of the environment is in controlling a stem cell’s behaviour.

It’s known that several extrinsic factors, such as neighbouring cells, chemicals in the environment, stress and oxygen levels influence stem cells. Understanding the pathways behind these interactions is of paramount importance in order to develop effective stem cell therapies, and might even lead to understanding disease, as in the case of cancer, where many of these pathways are switched off.

The stem cell niche concept must be mentioned here. In a tissue or organ, stem cells don’t roam around freely. They are found in specialised microenvironments, where stem cell growth and function is regulated and maintained.

The diagram above illustrates the concept. It is a place where stem cells can divide (1) or differentiate (2, 3). (4) is a special case: sometimes, a newly differentiated cell can re-enter the niche and become a stem cell again. But the most integral part of the niche are the niche cells, which control the environment by secreting chemicals, regulating oxygen levels and physical factors – all in order to make the stem cells feel comfortable. They regulate the renewal rate, control the stem cells’ ‘hibernation’ and when they do wake up, the niche cells tell them what to do.

We are still looking for many stem cell niches (in humans). We know bone marrow is a niche for hematopoietic stem cells (more specfically, the surface of trabecular bone). In the following picture, the ‘bulge’ area serves as the epidermal (skin) stem cell niche.

The intestinal stem cell niche is also known (ISC in the following diagram).

We know the fundamental principles behind how stem cell niches work, but we need to find out more exact details on the molecular interactions that go on there, the physical effect of the niche cells on the stem cells and basically how the niche cells manipulate the stem cells – so that we can copy their ways.

Another topic is cancer stem cells. This is another one of those hotly debated fields, with one side claiming that cancer stem cells are regular stem cells that have acquired mutations in its lifespan, while the other side claims that stem cells can be genetically predisposed to initiate cancer. The importance of this field is crystal clear: it can lead to novel cancer-fighting strategies (see diagram below). But there’s also a negative implication: if we mess around with stem cells by mutating them, we could be the cause of cancer.

The reason we can even hypothesise this is because stem cells and cancers share key characteristics, the most important being self-renewal. In stem cells, this is regulated, whereas in cancers, it is not. Figuring the control mechanism in stem cells could give us hints about what causes cancer, or just tips for a cure that stops tumour growth.

Since stem cells live for so long, there is a huge risk of accumulating genetic damage. As such, they need ways to maintain the integrity of their genome. Stem cell mutations are more dangerous than any other ones, since they will get passed on to the most daughter cells – this may be a tumour-starting process!

One source of mutation is reactive oxygen species (ROS), free radicals produced as a result of metabolism. Free radicals are the sluts of the chemical world, in that they will react with any chemical, including DNA. Stem (and other!) cells have developed ways to minimise ROS damage, such as detoxification enzymes (all controlled by gene expression). Another danger is the telomeres. Stem cells, like cancer cells, express telomerase, defending them from telomere erosion (but placing other risks which we don’t yet understand).

If a cell ends up being too damaged, ageing and cell death is the result. In stem cells, they can also show reduced self-renewal rates, reducing the repair rate of the involved tissue. In the worst case, cancer develops.

Stem cell therapy has been (rightly) hailed as the future of medicine. Cancer notwithstanding, some have seen the potential to cure blindness, diabetes and neurodegenerative diseases. But we haven’t reached that stage yet. Inducing stem cells is but the first step. We still do not know how to control them: inducing cardiac stem cells will not automatically regenerate a heart.

Case Study: Neurogenesis in Mammals

The brain of a mammal grows during its lifetime as a result of adult neuronal stem cells (NSCs). The whole process consists of adult neuronal stem cells dividing, differentiating, maturing and finally getting integrated into the brain’s circuitry. It is the basis of learning and memory.

That said, it’s only been confirmed to occur in two areas: the subventricular zone (SVZ) and the subgranular zone (SGZ) – see the picture below for an overview of neurogenesis in a rodent’s brain. All the coloured areas show places where neurogenesis takes place: the SGZ is in the dentate gyrus of the hippocampus, and is where some neuronal stem cells differentiate; those that originate in the SVZ move through the rostral migratory stream to the olfactory bulb, where they differentiate into granule and periglomerular neurons.

NSCs are multipotent; they can differentiate into all neural cells: neurons (the cells that transmit information), oligodendrocytes and ascocytes (making up the glia). One tricky thing about them is that while they can be retrieved from all areas of the nervous system, it’s only in the SVZ and the SGZ that neurogenesis has been observed, making those two areas the neurogenic niches.

I will not go into the details of the exact biomolecular controls of the NSCs (overkill), but I do want to bring up integration of the new neurons.

As can be seen in the diagram above, fresh neurons go through a specific developmental program, which varies depending on the neurogenic niche. Besides the set pathway, environmental clues play a large role in the maturation process. For example, depriving an organism of olfactory senses (smell) will prohibit the proper growth of olfactory neurons in the SVZ.

In fact, neuron cells can die as soon as they are four weeks old due to the organism’s experiences (i.e. amount of stimuli). This is the essential part of integration: these new neurons are not subjected to any different treatment, but go through the same biochemical control as the already-established neurons.

The implications of this are clear for the study of cognition, learning and behaviour, in that we have a clear link between the origin of new neurons, i.e. induced growth of an oft-used brain area, and tangible ethological consequences: banally put, an animal that uses its nose for everything will develop a brain capable of efficiently processing olfactory inputs. One noteworthy observation is that neurogenesis rate decreases with age.

This can all be studied using genetically-modified mice that produce too many, or too little, or defective neurons, and measuring their cognitive abilities using standardised tests.

You may be asking yourself about what can be achieved using this knowledge, and it’s important not to get carried away. We know that there are correlations between SVZ and SGZ neurogenesis and cognition, mood and various CNS disorders (notably epilepsy, strokes and Alzheimer’s). But what must always be kept in mind is that there may be other sites of neurogenesis we haven’t discovered yet! Correlation doesn’t imply causation – although in these cases, it’s probably more than simple coincidence. Nevertheless, we always must keep our feet on the ground, especially when messing around with the brain (granted, all this has been investigated only in rodent brains, not humans).

The basic fact behind all of these strategies is that a differentiated cell’s genome is no different from a stem cell’s genome – so they should both have the same potential. Of course, this doesn’t make the task easy, and all these processes are rather inefficient.

Nuclear transfer refers to removing the nucleus of an egg cell and injecting a somatic cell’s nucleus in its place. This egg cell can then be used to impregante an animal, giving rise to a clone , or it can be grown in culture as a source of embryonic stem cells (these are then referred to as SCNT: somatic cell nuclear transfer).

We can make a somatic cell – ES cell hybrid by cell fusion, giving us pluripotent cells. The mechanism behind this is not fully understood, but it does have something to do with the epigenetic state of the ES cell. However, it is generally not preferred due to its inefficiency and the resulting tetraploidy – making them diploid again is too risky (because of genome destabilisation).

Explantation refers to the culturing of multipotent cells (e.g. spermatogonial stem cells). Finally, we can customise viruses to infect somatic cells and induce pluripotency (the iPS cells I mentioned earlier are the products of this treatment).

At the end of the day, the way forward is to further our understanding of how our treatments work at the molecular and genetic level (see picture below).

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