Feature: Secrets of stem cell differentiation

How does an embryonic stem (ES) cell ‘know’ how to become one of the 200 plus adult cells into which it can transform? What does it need in terms of chemical, signalling and environmental cues to become, for example, blood, skin, nerve, heart or pancreas? Specifically, how do mesodermal cells become blood, heart or endothelium cells, and how endodermal cells become pancreas cells?

They are the kinds of question that occupy Professor Andrew Elefanty and Professor Ed Stanley, who jointly run a research group at the Monash Immunology and Stem Cell Laboratories in Melbourne. Their lab works on the biology and differentiation of pluripotent stem cells, a term that encompasses embryonic stem cells and the more recently developed induced pluripotent stem (iPS) cells. Both hold the tantalising promise of repairing and regenerating adult tissue in a range of disease and damage situations by virtue of their unique properties of pluripotency and self-renewal.

Most of Elefanty’s recent work involves human ES cells. The group’s major focus is in understanding how a human ES cell isolated from a blastocyst at the earliest possible stage of development can be directed down certain lineages of differentiation.

“Our approach and focus since about 2002 has been to generate novel tools and improve the technologies to enable our research into the biology of ES cells,” says Elefanty. At that time, the field of human ES cell research was in its infancy, and like the cells themselves, had to be grown step by step in the correct sequence for things to start working.

“This presented us with both a challenge and opportunity: to develop the best tools, reagents and protocols needed for successfully working with these cells. The main limitation was ignorance. Nobody really knew how to proceed, particular how to efficiently differentiate ES cells. So a lot of the protocols we developed along the way are still used by us and many others.”

The first step is the hardest

One of the team’s earliest and most significant achievements was working out the best way to grow and culture human ES cells and to do so on the scale needed for useful and reproducible differentiation experiments. As part of this, they developed novel culturing media that enabled the cells to head down different lineage pathways in a controlled way, as well as several new techniques to achieve specific differentiations such as blood, heart and pancreatic cell types.

“What we have come to realise, in fact, is that for all of the techniques we do and have developed, the preparation, culturing and maintenance of ES cells is of key importance for both being able to differentiate them well and for having them survive the development and differentiation process,” says Elefanty. “This was in one sense the most boring or tedious aspect of the process, but also ended up being the most critical – although not very exciting to talk about of course.”

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One of the major claims to fame for Elefanty and Stanley’s group is developing techniques that allow human ES cells to be genetically modified such that their differentiation pathway could be ‘tagged’.

A reporter gene, usually a fluorescent marker, is inserted into the cell genome at specific loci to highlight certain genes coming on or off. Live cells can then be monitored simply by microscopy to identify those cells heading towards the lineage of interest. Elefanty explained how this proved to be a very useful tool for improving the efficiency of in vitro cell differentiation and thus furthering their research program substantially.

“It also allows us to characterise the biology of the differentiating cells in ways that others can’t because we can use those reporters to isolate live cells that we want and grow just those cells for experiments.”

Late last year, the pair’s work in this area hit the headlines and the pages of Nature Methods when they reported a new modified human ES cell line, called ErythRED. These cells glow red when the globin gene, linked to a fluorescent tag, is switched on – that is, when the stem cells turn into mature red blood cells.

ErythRED will facilitate efforts to differentiate human ES cells into red blood cells in vitro for clinical applications and aid in identifying ES-derived red blood cells in transplanted animals. Elefanty said that in other similar work, the group could now identify several specific cell types derived from their ES cells such as mesoderm precursors, early heart cells, and cells that produce insulin and nerve cells. “We have tagged about 10 genes in all and have others in the pipeline,” he says.

In doing this work, Elefanty and colleagues have come to realise that, in most cases, no single gene is a perfect marker of differentiation and combinations of markers often are needed to identify a specified fate of human ES cells, which he describes as “notoriously difficult” when it comes to genetic alteration.

“In fact, we remain one of the few groups in the world able to do this consistently. There are usually a number of intermediate steps along the differentiation pathway that have to be reached and you need some way of marking the efficiency of each of those stages – this is partly why this whole process of stem cell research travels slowly.”

Fresh is best

Elefanty stresses the importance and increasing interest in persisting with human cells, even apart from the obvious direct therapeutic applications. “These cells are important for specific experiments such as lab-based models for investigating certain disease states when patient tissue is difficult to procure or as tools to screen potential therapeutic compounds for efficacy and toxicity.”

As an example, Elefanty described one of their ES-derived cell lines in which mature cardiac cells can be unequivocally identified by virtue of a genetically engineered marker. “We know that this cell line may be of great interest to the pharmaceutical industry because one of the mandatory requirements for most drugs is no adverse effects on the heart in terms of its beating or rhythm properties.

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“Current laboratory testing for this is sub-optimal because of the types of cells they use and there is a clear desire for better tools. Similarly, understanding fully how cells differentiate into blood cells or down pancreatic lineages and working out ways to make sufficiently large quantities for therapy would have a large market.”

In terms of the potential clinical uses of human ES cells, Elefanty explains there are basically four criteria or pillars that a stem cell has to satisfy to be of therapeutic value, and none have really progressed to an advanced-enough stage just yet. First, and probably foremost, the cell type has to be exactly the one it is supposed to be. Sounds like a trivial point, but many very smart people around the world are trying very hard to work out how their stems cells can reach that milestone.

Secondly, the cells have to be available in the scale needed for the purpose. Next, there are a few safety issues to be addressed to avoid cells in the mix that could grow into tumours or cause disease, and there has to be absolutely no risk of exposure to animal-type viruses or other harmful hangers-on.

Finally, therapeutic stem cells have to have a way of circumventing the body’s desire to reject foreign cells, and that in a way is the most obvious attraction of the iPS cells, which could potentially be reprogrammed from the patient’s own cells.

Elefanty predicts the area of ES cell use that will have the biggest impact on human health in the near future will involve stem cell-derived products for use in vitro.

“I think these sorts of uses will come first because if you are making cells to be used for testing drugs in the lab for instance, there are not the same constraints in terms of purity and safety. These applications are starting now as people make different cell types from stem cells, in particular heart cells, and liver cells, because many drugs are metabolised there and are toxic to the liver.

“Another area is making stem cells from patients with a particular illness to try to understand the abnormalities or pathology of the disease from a biological basis in the laboratory by studying and working with the stem cells. This is a very hot area at the moment.”

This feature appeared in the March/April 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.