From laboratory to bedside

"It is not that stem cell transplantation doesn't work, it is just that we need more work to figure it out." With this kind of simple optimism, and a little green jasmine tea, Professor Brent Reynolds chatted with Fiona Wylie about life, coincidence and the use of neural stem cells to treat spinal cord injury.

Brian Reynolds is one of a distinguished list of speakers making up a two-part session, "In the search for a cure for spinal cord injury - from laboratory to bedside", at the Australian Health & Medical Research Congress (AH&MRC) at the Melbourne Convention Centre from November 26 to December 1.

Reynolds moved from Canada to the Queensland Brain Institute (QBI) at the University of Queensland in 2004. His path to this point has been somewhat unorthodox to say the least, particularly for someone who published a Science paper and devised an important new tool for the entire field during his PhD.

Immediately after finishing his doctorate in 1994, Reynolds founded a company called NeuroSpheres, based on this new technology.

"I was the director of research and we worked with large pharma and several biotechnology companies to further develop and protect the technology," Reynolds says. "Today NeuroSphere transplantation technology is licensed to Stem Cell Inc, based in California, who are about to start clinical trials based on technology we developed and patented, which is kind of exciting."

Impressively, the technology is also the basis of Phase II trials by another company for treating stroke, and at least half a dozen clinical trials starting in 2007-2008.

The unorthodox route to the QBI began in 1997, when Reynolds opted out of science to study Chinese medicine. He and his family spent the next few years between Thailand, running a yoga centre, and Salt Spring Island off the west coast of Canada, where Reynolds had a Chinese medicine clinic.

The lure back to science came in 2002 when an old university friend in Vancouver, who was head of business development with a company called StemCell Technologies, contacted him because the company wanted to get into the neural stem cell field.

"Things weren't working, he heard that I had moved to the west coast so he asked if I would come and have a look at this stuff, and I started going to help him one day a week," Reynolds says. "It was also near a really good yoga teacher."

To help out, Reynolds got on the phone to former contacts looking for technology to license. One of these was Dr Rod Rietze, who used to work with Reynolds at NeuroSpheres. Rietze had just moved from Melbourne up to Queensland with Professor Perry Bartlett to set up the QBI.

It seems the next step was meant to be - Reynolds and his family had just been convinced by a friend from Thailand that Australia, and particularly Brisbane, was a great place to live.

"So Rod arrives and is telling me all about this new institute and says I should come and work with them in Brisbane, so it was perfect."

Neural stem cells

Since his arrival in the Sunshine State, Reynolds and his team at the QBI have been developing methodology (soon to be patented) to identify and expand distinct cell populations within a heterogeneous milieu of neural stem cells in culture to benefit transplantation therapies, in particular spinal cord injury.

"The existence of stem cells in the adult mammalian central nervous system (CNS) and our ability to isolate and expand them ex vivo provides a number of therapeutic opportunities when it comes to treating spinal cord injuries," Reynolds says.

Cell transplants into nervous tissue have been going on in animal models now for two to three decades. Primarily, most of the work has been done in rodent models of Parkinson' disease, with over 1000 reported studies of transplantation into the brain. Human studies have also been carried out, with 300 to 400 people receiving human foetal tissue.

Basically, there were lots of promising results, and some not so promising results. Reynolds uses this historical context to highlight the two main problems with neural cell transplantation, which he will discuss along with ways to solve these problems at the AH&MRC.

Firstly, there is never going to be enough primary foetal tissue available for transplants, especially given the ethical or moral issues to be considered, he says. The primary requirement in this field is therefore a renewable source of neural stem cells.

One possibility is embryonic stem (ES) cells, differentiated down the neural lineage and grown up in culture for transplants. The problem with ES cells is that they are undifferentiated cells to start with and there is a chance, even if only slight, that one of those cells doesn't terminally differentiate and grows to form a tumour after transplantation.

"All you are going to need is one tumour in one patient, and it will kill the whole field," he says. "That is what happened in the late '90s with gene therapy."

This issue is highlighted by a paper just published in Nature Medicine showing that human ES cells differentiated into dopamine neurons and transplanted into a rodent model of Parkinson's cured the symptoms of the disease. All of the animals, however, developed tumours. The solution is better ways to sort cell populations early on in the piece, and Reynolds' group is also working on assays to do this.

The other possible renewable source are neural stem cells grown as neurospheres, which are clusters of cells grown in tissue culture from primary neural stem cells isolated from either adult or foetal tissue. These neurospheres can then be grown up in large quantities in vitro for transplantation into patients.

As mentioned, Reynolds actually developed the neurosphere assay (NSA), which is now widely used to isolate, propagate and enumerate stem cells derived from the CNS. It is now recognised, however, that not all the neurospheres in a culture are derived from stem cells as first thought. About 90 per cent come from progenitor cells, so the numbers of stem cells represented by the NSA are largely indeterminate. Reynolds is also developing assays to address this problem.

Proliferation

The second major problem with growing neural stem cells as neurospheres is that only about 10 per cent of the cells turn into neurons. When the cells are given growth factors in culture to drive proliferation, it seems to push them predominantly down the astrocyte lineage (approximately 90 per cent).

Since generally only one to 10 per cent of transplanted cells survive, the numbers of cells needed for the treatment of one patient becomes unreasonably large.

"People have tried very hard and for a long time and push cells down the neural pathway and basically, it just does not work," he says.

Hence, a need existed for a more accurate way of determining and purifying precursors cells. "We have to know exactly what we are transplanting into patients."

Reynolds' team has come up with a new assay, called the neuroblast assay, which increases the number of neurons that are produced from neurospheres. These are then sorted to give a purity of about 90% neurons. The successful implementation of this technology also depends on being able to identify distinct population of cells within the heterogeneous population of stem and progenitor cells.

"We need to know exactly what is in the culture dish, and what each patient receives in a reproducible way."

Variable and indeterminate combinations of neuronal and other CNS cells are the most likely cause for the negative effects seen with those early transplants into Parkinson's patients. Part of the technology developed by the team at the QBI is focused on sorting the expanded cells to address this exact issue.

Ideally, they will be able to take stem cells from an adult donor, grow them up in tissue culture as neurospheres, sort out the neuronal and non-neuronal cell types, and then reproducibly transplant for each individual. The procedure will additionally allow controlled mixing of the sorted cells.

"You may not want to transplant all neurons - you may want to use 60 per cent neurons and 40% astrocytes. In fact, some transplant papers that report better results when neurons are transplanted with astroctyes."

The ultimate aim for this research is to have a renewable and defined source of neural stem cells that can be tailored for different patients to treat spinal cord injury, stroke, Parkinson's disease and more.

"Obviously this is just the first step, but we now have a way of figuring out what we need to."