The Royal Children's Hospital, Parkville, 3052 Victoria, Australia
Dr. Andrew Elefanty, Dr. Ed Stanley and Ms. Elizabeth Ng are researching the regulation of embryonic stem (ES) cell differentiation to mesodermal and endodermal lineages, as exemplified by blood, heart, endothelium, and pancreatic islet cells.
A major goal of their work is to realize some of the scientific and therapeutic potential that human pluripotent stem cells promise. These include unique opportunities for the study of early stages of human development, the generation of in vitro models for human diseases, testing of pharmaceuticals and other therapeutic products, and the production of transplantable cells for tissue repair and regeneration.
On Andrew, Ed and ElizabethAndrew: Elizabeth and I study blood cell differentiation, and Ed's laboratory maintains a major interest in pancreatic endoderm differentiation. Elizabeth also focuses on the development of differentiation media and methods, including the spin embryoid body and APEL medium platform that forms the basis of our differentiation protocols. Ed's expertise in vector design has been instrumental in our successful generation of fluorescent reporter cell lines in human ES cells by homologous recombination.
Ed, Elizabeth and I were working in the same laboratory at the Walter and Eliza Hall Institute of Medical Research when James Thomson and, shortly afterwards, Martin Pera published the generation of human ES cells in 1998 and 2000. The three of us felt that this was a really important avenue of research to pursue, so we moved to Monash University, where Martin Pera had his laboratory, to start working with human ES cells.
On PSC Research
What are the biggest advances you think the field will achieve in the next 5 years?
Ed: To quote Malcolm McClaren, “an idea shared is an idea lost.”
Andrew: So we’re not going to tell you! More seriously though, the next development in our field will probably be the generation of populations of cells that will be of clinical benefit. It’s just starting now, with trials of retinal pigment epithelial cells, and neuronal cells, and we think that pancreatic endodermic progenitors are not far away.
I think that it will be interesting to watch the sequence in which cell types come to the clinic. In my opinion, it will be driven by pragmatism: the earliest cell therapies will employ whichever happens to be the easiest cell type to make in sufficient quantities, and will be restricted to the kinds of cells that will be functional without the requirement to build a complex 3D organ structure. The next challenge would be to try to build bio-replicas of organ structures or maybe even to design new functional organs that don’t look anything like their endogenous counterparts.
Ed: I’m very guarded about the immediate impact that stem cell technology will have on the treatment of the common conditions that ail modern Western society. Many of the illnesses that people are trying to cure or improve treatments for are age-associated or caused by overconsumption. In the treatment of cancer, for example, the main impact that I see for stem cells will be as a tool to study the process and test new therapeutic agents. And even for cardiovascular disease, stem cell therapies are not really going to help that much if everyone’s still smoking and eating hamburgers 24 hours a day! I think the area where the technology has a potential effect is where something simple has gone wrong. In my own area of research, type I diabetes might turn out to be just that type of disease, but it may also turn out to be a much more difficult nut to crack.
Andrew: Another way of looking at it is that conditions in which there’s a simple deficiency of a cell type will lend themselves to being treated by the in vitro generation of lineage-specific precursors. Stem cells could also contribute to the screening of therapeutic agents and understanding pathogenesis, or determining new treatments. There has been intense activity in making induced pluripotent stem (iPS) cells from patients suffering from a range of illnesses in order to be able to mimic those diseases in a dish, but of course, the next stage will be how you use those tools to find out something new. That will be quite a challenge as well.
On PSC Culture Techniques
1. What are some challenges in differentiation of hematopoietic cells from PSCs, technical or otherwise? (e.g. variability in reagents, primitive vs. adult hematopoietic stem cells (HSCs), bias towards a particular HSC colony type)
Andrew: I think that the greatest difficulty that we have in generating hematopoietic stem cells from pluripotent stem cells (PSCs) is that we do not know the exact sequence of developmental steps that are required to generate the first transplantable HSCs from the aorta-gonad-mesonephros (AGM) region prior to colonization of the fetal liver. Therefore, we are not sure how to replicate this process for differentiating PSCs in vitro.
2. There's been a trend in the pluripotent maintenance field, moving from FBS and feeder conditions to increasingly defined and now serum-free media. What are your thoughts on this?
Andrew: We try every new method that comes out, because we want to get away from the variability of feeder systems and knockout serum replacement (KOSR). We are always optimistic, but the problem is that people who develop these new media obviously develop them in the context of their labs, where things are finely balanced and they work – and then the media tend not to be robustly applicable enough to survive 'in the wild'.
Ed: One thing that has always puzzled us is that there are a lot of substrates out there that people have published for stem cell culture (like laminin or fibronectin). You have to think though, that one substrate by itself is not likely to work that well, and so one logical solution might be to combine these purified or defined substrates together. What we’d like to know is, is it simply intellectual property constraints that have stopped that from happening? In other words, is it too difficult or not cost effective for one company to license a number of individual components? Or is it that the chemistry involved in the generation and combination of these elements is not straightforward?
Elizabeth: One other thing we’ve spent quite a bit of time looking at, even though it seems a little boring, is comparing different dissociation methods, both for the ES cells and for embryoid bodies differentiated from those cells. In our experience, each of the different dissociation agents seems to have different pros and cons. For example, with non-enzymatic dissociations based on EDTA, you get the best retention of surface epitopes, but lousy survival. All those considerations are annoying subtleties of the system that influence how you try to do things, and probably influence the outcomes as well.
1. One of the points we talk about at STEMCELL is that there’s a fair bit of work that goes into making feeders and then qualifying them. Is that a point that resonates with you?
Andrew: Absolutely. We are always desperate to get away from feeders. But we know that the cells on mTeSR™1 are slightly different from cells grown on feeders – they don’t look exactly the same.
Elizabeth: And they are chemically not the same. That is, if you look at the cells using vibrational spectroscopy, their 'signature' differs from PSCs grown on feeders. Also, as we said, we have difficulty making our spin embryoid bodies from mTeSR1™ cultured PSCs.
2. What applications do you use mTeSR™1 for?
Andrew: We use mTeSR™1 for example, when we want a monolayer (two-dimensional) differentiation culture free of feeders. We’ve used it for cardiomyocyte differentiation, with as our differentiation medium.
Elizabeth: When we wish to look at early stages of mesoderm or endoderm differentiation, we can form nice discrete PSC colonies in monolayer cultures in mTeSR™1 with our MIXL1-GFP reporter line. We can then observe individual cells and groups of cells within colonies as they undergo the epithelial-mesenchymal transition that marks the transition to a primitive streak-like cell in the culture dish. We can correlate this morphological process in live cells with the appearance of GFP, reflecting expression of the MIXL1 homeobox gene that marks the primitive streak in the embryo. That is quite an interesting application if you’re interested in the details of early differentiation and patterning. The advantage over feeders is that you can see the small colonies better, and you can watch the differentiation happening in each colony at the same time.
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