Sydney researchers zero in on stem cells

Researchers at the Diabetes Transplant Unit at the Prince of Wales Hospital in Sydney have developed a new technique for extracting single human embryonic stem (ES) cells, bringing them one step closer to harnessing the therapeutic potential of stem cells.

Human ES cells are derived from the inner cell mass of four or five day old blastocysts, which arise from spare fertilised eggs. Made up of several hundred cells, this heterogeneous population of ES cells is cultured into a colony.

The resulting ES cells have the potential to become any cell type found in the body and if allowed to differentiate will form a mass of mixed tissues, such as muscle cells or neural cells.

Dr Kuldip Sidhu has devised a technique using flow cytometry, or fluorescence activated cell sorting (FACS), for extracting and cloning single cells from these heterogeneous colonies. These single cells then form a homogeneous colony of ES cells.

"This homogeneity gives us more control in defining a cell lineage of interest such as the formation of insulin producing cells," said Sidhu.

Sidhu said it has taken some time to optimise the technology, as it is very difficult to resuspend a single cell and keep it alive.

"In our hands, at the moment, 95 per cent of these cells are viable once we resuspend them and look at them under the microscope," he said.

Sidhu said the technology is unique in selecting cells using FACS sorting, which is a very accurate way of looking at single cells.

"There is an Israel paper on the technique of isolating single ES cells from a colony, but this involved the researcher physically and subjectively picking up the cells under a microscope," said Sidhu. "When I repeated this technique and then progressed to validate it, I found that I picked up groups of cells. However, using the flow cytometry machine enables single cells to be picked up."

The researchers now have three single ES cells clones growing on a human fetal feeder layer of cells. Based on gene expression studies, one of these clones is tending towards an endodermal cell phenotype, which would progress to form the gut and liver.

"It's early days yet, and we need to understand the clones better before we can recognise their full potential," said Sidhu. "But we are one step closer to harnessing the potential of stem cells down the track and this technology will go a long way towards future therapies."

The US Juvenile Diabetes Research Foundation recently awarded a US$140,000 grant to Sidhu and Prof Bernie Tuch to continue the research.