Feature: Stem cells, made to order

The appearance of Dolly the sheep in 1997 sent shockwaves through the general public and the scientific community alike. A living clone and a clear demonstration of the power of somatic cell nuclear transfer (SCNT), which involves replacing the nucleus in an oocyte – a female germ cell – with a nucleus from an adult somatic cell and triggering development.

The result is a reprogramming of the nucleus from the original differentiated somatic cell into a totipotent stem cell, which has the potential to form an entirely new animal, as it did in the case of Dolly, spawned as she was from a mammary cell.

Alternatively, instead of producing an adult clone, embryonic stem cells can be harvested from the blastocyst – cells that will be genetically compatible with the somatic nucleus donor.

One of those who was particularly excited by Dolly was stem cell veteran Dr Paul Verma, now at the Monash Institute of Medical Research. Verma had long been interested in cellular reprogramming, dating back to his research in knocking out genes in pigs for organ transplantation in the 1990s. But it was nuclear transfer that most captured his imagination.

“I was fascinated by the process,” says Verma. “How could you take an adult cell and make it go backwards in differentiation? The dogma I was taught in university was that once you got to a certain stage of development, you couldn’t go back. But when Dolly was produced, it changed the way I saw the world. It made me question everything.”

Then, in 2006, another milestone was reached: the production of the first induced pluripotent stem cells (iPS cells). Like SCNT, iPS cells start with an adult somatic cell, but a number of key transcription factors trick the cell into reverting back to a pluripotent state that closely resembles embryonic stem cells.

Neither technique is perfect at this stage, says Verma. SCNT is the more established approach, but it’s inefficient; it took nuclear transfer to 440 oocytes, to produce Dolly.

It also has a tendency to produce phenotypic abnormalities in cloned animals and is plagued by ethical concerns relating to the source of the oocytes, specifically in humans. Speaking of which, no-one has been able to get viable embryonic stem cells using SCNT in humans to date.

Induced pluripotency avoids the ethical issues, but runs into problems when it comes to the reprogramming of the adult cell, which is typically done with an integrative virus. However, the transcription is often incomplete or corrupted by viral integration. IPS cells also appear to be more prone to form tumours, limiting their use to the lab rather than the clinic until these issues are resolved.

One of the focuses of Verma’s lab is to overcome some of these limitation with iPS cells. “We’re working on the one hand to try to find the most efficient way to make iPS cells,” he says.

“And on the other hand we’re looking at a number of other approaches to making these to be DNA non-modified iPS cells. When the two come together, that will give us a robust outcome.”

Verma’s lab has focused on embryonic stem cells from many species for the last several years, and is one of the most practiced in the country. “We have a lot of experience of deriving embryonic stem cells from livestock, from mice and in culturing human embryonic stem cells. Having that hands on ability was critical in this sort of research,” says Verma.

“Every embryo has an embryonic stem cell in it, yet not every lab is able to produce a stem cell line. It really boils down to expertise and culture conditions. You need to be able to recover the stem cells from the embryo and then maintain them.”

They also streamlined the process for producing livestock iPS cells to make it more efficient and less prone to error. One of the steps they improved was the viral system used to transfect genes into the somatic cell.

“People have used lentiviruses and retroviruses, but we’ve used a pantropic virus, which infects more cells than a lentivirus. As a result, we know which parameters are critical to get success, and we’ve fine tuned those parameters so we can relax other ones.”