Clonal blastocysts are the real deal

The fertility clinic that provided the oocytes from which Dr Andrew French created the world's first authentic cloned human blastocysts by somatic cell nuclear transfer (SCNT) is just over three metres away, on the other side of the wall in his California laboratory.

Those three metres, and a few precious minutes, may have measured the difference between French's success and the failures that have frustrated the best efforts of stem cell researchers around the world to create individualised embryonic stem cells (ESCs) by SCNT, for therapeutic applications and research.

In January, French's team at private stem-cell company Stemagen, in La Jolla, announced in the on-line version of Stem Cell Express that it has achieved a feat that has eluded some of the world's leading stem cell researchers for at least half a decade.

The Australian stem cell scientist's team inserted the nucleus of a diploid somatic cell into an oocyte stripped of its nucleus, then induced it to divide parthogenetically to form a blastocyst - the precursor to a human embryo.

The tiny, berry-like clusters of undifferentiated cells wrap around a core of totipotent embryonic stem cells that have the capacity to differentiate into new ESCs, or any one of the 210-odd specialised cell types that form the human body.

Why did Stemagen succeed, where others had failed?

French believes his team's ready access to oocytes from highly fertile young women may have been an important factor, but also says the team had painstakingly refined its techniques through each iteration. He says they paid particular attention to removing the complete contents of the original haploid nuclei in each oocyte, to ensure there were no chromosome fragments to interfere with the replication of the donor nucleus.

Their meticulous approach worked: five SCNT-derived blastocysts from only 27 oocytes is an unprecedented success rate in a field notorious for meagre success and a very high oocyte wastage rate.

Stemagen recruited its Australian chief scientific officer from Monash University's Centre for Reproduction and Development, where he led a research program to develop transgenic cattle, and transgenic animals for laboratory research. He maintains close links with the centre.

French's team produced five SCNT-derived blastocysts over a period of about eight months, and each time had to make what he describes as a "heartbreaking" decision to hand the blastocysts over to an independent company to carry out genetic tests to provide absolute proof that they carried the somatic cell donor's genes.

"It was a fairly long and involved process, because it involved a number of independent groups looking at the data to [show] we have indeed produced clonal blastocysts," French says. "As each result came out, we became more convinced that we were seeing the real thing - it was a very exciting time.

"The SCNT field for human embryos has had some setbacks, and we had to be very clean up front and scientifically rigorous if we were to get the independent verification we needed to ensure we could get it through peer review and into publication.

"We were obviously optimising our procedures along the way, and it has given use the confidence to move forward."

---PB--- Scientific rigour

With the controversy, disappointments and spurious claims that have surrounded SCNT research, the peer review process required nothing less than absolute scientific rigour.

In 2004 South Korean scientist Woo Suk Hwang claimed to have created the world's first cloned human embryos by SCNT, and produced stem cell lines from them. Overnight, Hwang became a national hero, only to retreat in disgrace after confessing his results were fraudulent.

Miodrag Stojkovic's research group at the University of Newcastle in the UK reported in May, 2005, that they had created three cloned blastocysts, but were unable to reproduce an embryonic stem cell line.

In a news article in the January 17 issue of Nature, Stojkovic was quoted as describing the Stemagen advance as "a huge difference" from what his own team had achieved.

Stojkovic, now at the Cellular Reprogramming Laboratory at Prince Felipe Research Centre in Valencia, Spain, is an associate editor of Stem Cells. He congratulated French's team for submitting their blastocysts to genetic analysis, and said there was no doubt that at least one blastocyst was a genuine clone.

But Robert Lanza, from Stemagen's competitor Advanced Cell Technology in Los Angeles, said the article in Stem Cells lacked data needed to confirm the cells were fully reprogrammed to an embryonic state, and that the blastocysts were in good condition.

He observed, from the published photographs of the blastocysts, that they looked "very unhealthy".

French told ALS: "We think the quality of the blastocysts improved each time, and we've had expert advice that the real challenge is to induce a SCNT oocyte to produce a blastocyst - once you're past that stage, it doesn't matter how the blastocyst looks, you have a good chance of getting embryonic stem cells from it."

The five blastocysts were the final yield from 29 experiments over eight months to implant a donor nucleus in surplus, enucleated oocytes obtained from young women between the ages of 20 and 30 after hormonal treatment to induce superovulation.

The Stemagen researchers sacrificed the opportunity to provide ultimate proof-of-concept of one of the most anticipated advances in stem-cell research: isolating embryonic stem cells from the blastocysts and expanding them into self-perpetuating clonal colonies.

French says Stemagen is now working to create more SCNT blastocysts with the aim of isolating and culturing embryonic stem cell lines for research into new therapies for genetic diseases and toxicology screening for new drugs.

None of the techniques his team has used are patentable, so Stemagen plans to capitalise on its success by providing cell lines to other research institutions or companies developing new drugs or therapies for inherited disorders.

"The major benefits we expect will come in the area of using SCNT to create cell lines from individuals with specific inherited disorders," he says.

---PB--- Accessory genes

French says the advantage over the standard approach of creating mutant cell lines or transgenic animals with a particular type of defect to study a disorder, or to test new therapies, is that with SCNT, the mutation comes as just one component of the individual's complete genome - with any "accessory" genes that contribute to the disorder, and the individual subject's complete complement of micro-RNA regulatory elements.

"You get everything," he says. "So you can change anything in the cell to see what happens.

"For example, if someone is affected by Alzheimer's disease, you can make a representative ESC line and use it to screen thousands of molecules that might have a therapeutic benefit.

"You can also go into an ESC and repair a defective gene, and then implant it into the subject."

ESCs with "repaired" genes might be a better option than transgenic haemopoietic stem cells for treating blood disorders such as thalassaemia or sickle-cell anaemia, or immune-system defects, he says.

Haemopoietic stem cells are already partially differentiated, and while they can differentiate into any blood or immune-system cell, there is some evidence that they require the support of other, non-haemopoietic cell lineages to function efficiently. Cell communities in effect create their own niches with "friends" that supply the growth factors and signalling molecules needed to renew themselves.

ESC cells could also differentiate into non-haemopoietic lineages to recreate the full suite of cells and replace these cells, as well as all blood and immune system cells, he says.

Some people might also want to consider the possibility of preserving some of their own stem cell lines as an insurance policy against any future life-threatening disorders, such as heart disease.

French says Stemagen is already receiving inquiries for SCNT-derived embryonic cell lines for research, including from Australia and New Zealand.

Australian stem cell researches appear to be concentrating on downstream applications of SCNT, such as therapies for brain disorders like motor neuron disease, Alzheimer's disease and Parkinson's disease.

"We're at a stage in stem cell therapy equivalent to the penicillin era of antibiotics, and there are opportunities in Australasia to work with different groups, working with different genetics," he says.