Do-it-yourself stem cells

Take ordinary skin cells from a Parkinson’s disease patient and use modified retroviruses to insert four genes that will reprogram the cells to revert to pluripotency.

With a cocktail of cell-growth factors, reshape the new stem cells into neurons like those that populate the substantia nigra, the mid-brain factory for the neurotransmitter dopamine.

Now fast-forward the ageing process and use microarray technology to determine how patterns of gene activity in the Parkinsonian cells diverge from those of normal cells.

For the first time, you might see Parkinsonian cells form Lewy bodies – toxic clusters of alpha-synuclein protein, implicated in the genocide of dopaminergic neurons.

Molecular geneticists have dreamed of inducing ordinary skin fibroblasts to revert to a fully pluripotent state, effectively recreating the embryonic stem cells from which the fibroblasts arose.

In 2006 Professor Shinya Yamanaka’s research team at Kyoto University turned mouse fibroblasts into new stem cells, virtually indistinguishable from embryonic stem cells, which they termed induced pluripotent stem cells (iPS cells).

Yamanaka’s team created iPS cells from human adult fibroblasts in 2007; Professor James Thomson’s team at the University of Wisconsin, Madison, achieved the same feat independently, to share priority.

Around 11 months later, Dr Paul Verma’s research group at the Monash Institute for Medical Research in Melbourne has done the same with mouse and human fibroblasts.

Their achievement is enormously significant: until this year, Verma’s group and other Australian stem-cell researchers relied on the largesse of Yamanaka and Thomson to obtain iPS cells for research.

With a home-grown source of iPS cells, a world of possibility opens up for Verma’s team, and for Australian stem cell researchers in general. This is as good as it gets: iPS cells are an ideal tool for exploring how genetic errors combine with environmental influences to cause inherited metabolic disorders.

“We wanted to develop our own iPS cells lines so we could use the technology here in Australia, to study the diseases that interested us,” Verma says.

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Freshly minted copies

Just a few years ago, somatic cell nuclear transfer (SCNT) was hailed as the technology that would transform research into how inherited defects derange cell function.

SCNT is technically challenging: it requires an enucleated oocyte to be implanted with the nucleus of a fibroblast or other adult cell from someone with the hereditary disorder of interest.

The result is less than perfect. SCNT-created stem cells have not yet been derived from humans, and animal studies have shown SCNT-stem cells retain the donated oocyte’s mitochondrial genes.

In contrast, iPS cells are freshly minted, reprogrammed copies of the patient’s nuclear genome, complete with matching mitochondria, which will allow researchers to investigate the special class of hereditary disorders involving mitochondrial mutations.

Verma’s team’s first use of its do-it-yourself iPS cells will be in studying why pancreatic islet cells come under assault from immune system, causing type 1 diabetes.

They will also collaborate with University of Melbourne researchers to determine how trinucleotide expansion repeats in the frataxin gene trigger the progressive loss of spinal cord sensory neurons in the crippling hereditary disorder Friedreich’s ataxia.

“We now have the ability to look at some of the rarer diseases that involve the interaction of multiple genes in disorders that manifest only as people age – cardiac disease is also obviously a major focus,” Verma says.

His team employed the same methodology as Yamanaka and Thomson: a retrovirus-delivered cocktail of four genes required to maintain stem cells in an undifferentiated, pluripotent state.

The genes are: the c-Myc oncogene, a regulator of cell division; the SOX-2 transcription-factor gene and the Octamer-4 (Oct4) homeodomain transcription factor gene; and the Kruppel-like Factor (KLF-4), a regulator of c-Myc induced apoptosis.

Verma says his team wants to improve the efficiency of the technique, and find alternatives to the retroviral vector system. Typically, fewer than one in a thousand cells (1:1000 to 1:10,000) reverts to pluripotency, and while the iPS cells seem to have all the properties of embryonic stem cells, they differ subtly because there is no way to control the copy number, or where the retroviruses insert the transgene “cassette” in the host cell’s chromosomes.

Insertion at the wrong locus can disrupt the activity of nearby genes, which might explain why all but a tiny minority of cells fail to revert to pluripotency.

“We can now produce embryonic stem cell-like lines predisposed to the diseases we are interested in – for example, we can produce cardiomyocytes to study heart disease, or neurons to study complex neurodegenerative disorders like Parkinson’s.

“A lot of these disorders involve age-related effects that can be replicated by accelerated aging in culture, but the environmental contributions are more difficult to reproduce.

“That’s not necessarily a bad thing, because if we have the right cell line, and we have some idea of what the causative environmental effects might be, we can experimentally introduce them into the cell culture.”

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Eliminate the oncogene

The disease-prone cells can also be used to test experimental drugs that might alleviate the disorders in question, Verma says.

Non-biological delivery systems, like the layered double hydroxide (LDH) nanoparticles being developed by Professor Max Lu’s team at the Australian Institute for Nanotechnology and Bioengineering in Brisbane, may be an alternative for transporting messenger RNAs into cells to achieve transient synthesis of the pluripotency factors, without inserting the genes permanently into the host cell nucleus.

The iPS cell technique is implemented to generate mouse models of genetically complex disorders, via the usual intermediate step of creating chimaeric embryos by combining iPS cells with early embryos.

By selecting and mating chimaeric mice whose ovaries or testes are formed from iPS cells, researchers can produce pure breeding lines predisposed to the disease inherent in the original iPS cells.

But the presence of the c-Myc oncogene in the package of reprogramming genes also predisposes the mice to a variety of tumours associated with c-Myc mutations, so Verma’s team hopes to eliminate the oncogene from the pluripotency package.

Dispensing with retroviral vectors is another challenge. Verma says overseas researchers have induced reversion to pluripotency with adenoviruses, which do not integrate their genes in the host-cell’s genome.

In September, the NSW and Victorian governments funded the Verma laboratory and colleagues in Sydney to produce human iPS cells. “They’ll be happy with their investment, because it took us only around four months,” he says.

The ultimate aim is to identify how mutations derange normal cell function, then perform personalised gene therapy with iPS cells made from the patient’s own fibroblasts, after correcting the inherited genetic error.