Feature: At the frontier of stem cell research

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

The world of cellular differentiation has been shaken up over the past few years with the discovery that the differentiation of adult cells is not a one way street. The developmental fate of cells was once thought to be determined when they began travelling down the road of differentiation, but research has shown that the identity of adult cells can be manipulated, opening intriguing prospects of using cellular reprogramming for therapeutic purposes.

Professor Melissa Little and her team from the Institute for Molecular Bioscience (IMB) at the University of Queensland in Brisbane are at the forefront of cell dedifferentiation and are working on inducing mature kidney cells back in developmental time to a specific kidney stem cell stage.

“Ultimately, our aim is to generate kidney stem cells in vitro for use in bioengineering to create replacement organs or to develop the stem cells in situ and encourage a portion of the kidney to start nephrogenesis again and thus regenerate in a living animal,” says Little. Alongside this cellular reprogramming work, Little’s team is also working on pushing human embryonic stem cells along the pathway to becoming differentiated kidney cells.

Dedifferentiating adult cells

Induced pluripotent stem cells (iPSCs) are produced by ‘inducing’ adult cells back to an embryonic stem cell-like state through genetical reprogramming. It turns out the epigenomes of differentiated cells are surprisingly plastic, meaning iPSC lines are turning out to be remarkably easy to generate. Those working in the induced pluripotency field are now producing cells at various stages of development along the path between embryonic and adult cells using novel reprogramming techniques.

Although, at this stage, only a few cells have been so reprogrammed, the process has proven highly reliable. Typically, it involves introducing a combination of specific transcription-factor genes into cells to produce many extra copies of the transcription factors. After some time – days or weeks – dormant embryonic or developmental genes can be awakened and begin to stably re-express proteins that reprogram the cell to become pluripotent or dedifferentiate to a particular cell type.

As a developmental biologist, Little’s research has focused on the molecular genetics of kidney development and the causes of renal disease. Her team is acknowledged internationally for its work in defining the genes involved in normal kidney development and integrating this knowledge with understanding how the adult kidney responds to damage.

“The kidney is not a highly regenerative organ. It is thought that it contains no stem cells or that they cannot be harvested from, or successfully delivered to, end-stage renal patients,” says Little. “Mesenchymal stem cells do exist, but they do not become nephrons, they appear to support the nephrons.”

The incredible intricacy of the kidney may partly explain why it does not have a high regenerative capacity. Each nephron – the workhorse of the kidney, performing the key functions of waste elimination and water regulation – contains numerous different cell types arranged in a distinctive order along its length. And the one to three million nephrons that a human kidney contains are, in turn, tightly organised.

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The mammalian kidney derives from mesenchymal tissue in the embryo, as does the blood, pancreas and heart. The initially disorganised mesenchymal cells of the developing kidney undergo a transition and become organised into functional epithelial tubules or nephrons. The number of nephrons in the kidney is determined in utero – by 36 weeks gestation all of the nephrons have formed.

Chronic kidney disease is on the increase in Australia, rising by around six per cent each year. This means that an ever-increasing number of people are dealing with end-stage renal failure and there are limited options for the treatment of this condition, not to mention the demands this places on the health system. Kidney disease is also very prominent in transitional populations, such as indigenous Australians. “These populations have undergone huge changes in diet and lifestyle over a relatively short period of time, which may explain these high disease rates,” says Little. “Hypertension and diabetes are also linked with chronic kidney disease.”

People with chronic kidney failure can manage with 30 per cent of their kidney function but when this drops to 10 per cent they need to go on dialysis. However, there is growing recognition that the disease state arising from renal failure results from more than just the loss of blood volume regulation, small solute and toxin clearance that dialysis therapy replaces.

Despite the risk of infection and rejection, the gold standard for the treatment of chronic kidney disease is still a kidney transplant. “At the moment dialysis is the primary treatment option for people with chronic kidney disease,” says Little. “Only one in four patients receives a transplant.”

Because of the shortage of kidneys for transplant and the limitations of dialysis, there is a need to find other ways to treat kidney disease. Little is interested in understanding how to produce the precursors to adult kidney cells and thus find options for regenerating the kidney.

A top down approach

One of the two approaches Little’s team is using in their quest to regenerate kidney cells is dedifferentiation. This involves taking adult nephron tubule cells and coercing them into reverting to their mesenchymal developmental state.

“We know stem cells give rise to tubular cells, so we are taking nephron tubular cells back to a ‘parent’ cell, or nephron progenitor-cell state, and then developing new nephron cells from these progenitors,” says Little. “These new nephron cells could potentially become one of the many different cell types that make up a nephron.”

Little’s team is working with two cell lines: an immortal human kidney tubular cell line and freshly isolated kidney tubular epithelial cells from mice. They have assessed 20–25 transcription factor genes they know are critical for making the progenitor cell state and have forcefully expressed these genes in lentiviral vectors with which they infect the cells. The lentivirus integration is stable and, unlike some vectors, lentiviruses do not require cells to be dividing for them to enter, they can easily get into quiescent cells. The lentiviruses integrate permanently with the cells’ genomes, which then begin expressing the genes they are carrying. The products of the transcription factor genes then turn on genes of interest. Over time the cells become stable and committed to their induced progenitor-cell state.

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“The cells need to divide for a while to make the different proteins,” says Little. “This takes about a month in culture because the genes need to be stably activated for about 10 days. If you force a cell to make different proteins the cell needs the changed environment for a while for it to have stable gene and protein expression.”

When cells are closer in phenotype it is easier to get them to turn into each other. Recent work in the pancreas successfully dedifferentiated a pancreatic exocrine cell into an insulin-producing beta cell by manipulating three transcription factor genes. Both these cell types share a common progenitor during embryogenesis, which made this process relatively easy.

The dedifferentiation technique has also been used successfully in blood for a number of years. Again, these cell types are closely related and the work occurs with small changes close to the differentiation-end of cell maturation. Little suggests turning a kidney cell into a progenitor cell state is likely to be harder than this.

“We are taking terminally differentiated kidney cells and pushing them back to a progenitor cell state, which is challenging because the changes we want to induce are quite large,” she says. “Although this should be easier than taking the cells right back to the embryonic stem cell state.”

At this stage the work is very experimental, and Little says they are not concerned about issues such as uncontrollable cell division or the fact that the US Food and Drug Administration would not allow permanent lentiviral changes to be made to cells proposed for therapeutic use.

Currently, the research is focused on identifying what genes will induce dedifferentiation in the cells. “At the moment we are working on getting a baseline in the in vitro work. Once this is established we plan to move to in vivo studies,” says Little. She recently received an NHMRC project grant that will fund this work for the next three years.

Because of the rapid progress that is occurring in the inducible pluripotent field, many approaches have been devised for integrating genes into cells. It can now be done without the involvement of viral integration by using proteins that are directly taken up by cells.

Little suspects that there might be microRNAs and other regulatory factors involved in turning genes on, but because more is known about transcription factors this is what they are working with at the moment. Her team has almost completed screening the set of transcription factor genes they have identified as essential for dedifferentiation to occur in kidney cells.

“We have successfully identified a number of combinations of genes that look like they will do the job and in future we could inject these into mice to look at things in vivo,” Little says. “We have got the duck, now we need to find out whether it quacks.”

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A bottom up approach

The second approach Little’s team is using involves pushing inducible pluripotent stem cells or human embryonic stem cells towards becoming differentiated nephron cells. This work is being conducted in collaboration with Professors Andrew Elefanty and Ed Stanley at Monash Immunology and Stem Cell Laboratories in Melbourne, whose team are experts in human embryonic stem cell culture (see page 58). “It is the only group in the world to have developed a number of human embryonic stem cell lines with fluorescent tags driven by specific gene promoters,” says Little.

This work has generated lines of embryonic stem cells that produce fluorescent proteins at certain stages in differentiation. One of these is a cell-line that turns green when it reaches a certain stage in mesodermal differentiation. This is important for blood, heart and kidney development. The team is then adding on two additional colours – red and blue – to show when the cells take the next steps towards developing into kidney cells in particular.

“Ultimately, we will have a cell line that turns green then red then blue,” says Little. “Screening for fluorescence is easier than doing it blind and looking for gene expression. It enables us to more easily identify the cell type we have, and then we can double check that these cells express the genes for that stage of development.”

Little has begun the screening for novel compounds that will induce the initial cell lines to turn green. This work is a collaboration with Professor Robert Capon, also at the IMB, who has created a large collection of novel chemical compounds acquired from marine species around Australia.

The project is an ambitious one, but is another example of the frontier mentality of Australian researchers in stem cell biology. “These are difficult and challenging techniques and are not used widely,” says Little. “We are pushing boundaries.”

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