When the first human embryonic stem (ES) cell line was established in 1998, people had high expectations that regenerative medicine would soon become a reality. With the ability to differentiate into any type of cell, ES cells could help restore lost or damaged organs, even neurons in the brain. Amidst much-publicized ethical concerns surrounding the use of ES cells early in the new century, scientists in 2006 discovered a means to reprogram adult cells into ‘all-purpose’ stem cells known as induced pluripotent stem (iPS) cells. At the time, iPS cells looked like the silver bullet that would obviate the ethical concerns that had previously blocked stem-cell research and lead to a cascade of therapeutic applications.
Almost five years later, it is now recognized that there is still much to be proved before stem-cell therapies can be brought to the clinic. “The initial enthusiasm has passed,” says Davor Solter, professor and senior principal investigator of the Mammalian Development group at the A*STAR Institute of Medical Biology (IMB). “Our expectations for stem cells now have more reasonable proportions.”
In Singapore, a country known for its strong commitment to stem-cell science, research is diversifying more than ever. About 20 groups at various A*STAR institutions are working with a variety of objectives, from establishing ES cell lines to developing bioprocess technologies and utilizing iPS cells to study disease. A*STAR has pledged its continuing support to stem-cell research through relatively liberal regulations and ample public grants. In the past five years, A*STAR has allocated S$110 million to its Singapore Stem Cell Consortium.
Alan Colman, who contributed to the generation of Dolly the sheep, now uses iPS cells to model a rare aging disease, Hutchinson–Gilford progeria syndrome.
Modeling disease
Much attention and hope have been invested in iPS cells. The potential of iPS cells as a substitute for ES cells fascinates many well-established stem-cell researchers, and competition has been heating up to create safer and more useful iPS cell lines. “An iPS cell is very similar to an ES cell, but easier to make. We’ve found iPS cells to be very useful for modeling certain diseases,” says Alan Colman, principal investigator of the Stem Cell Disease Models laboratory at the IMB.
Colman is known for his contribution to the ‘birth’ of Dolly, the world’s first mammal to be cloned from an adult somatic cell. Colman’s contribution to the project occurred in 1996 when he was a research director of PPL Therapeutics in the UK. At that time, his team collaborated with scientists at the neighboring Roslin Institute to further evaluate the somatic cell nuclear transfer (SCNT) technology, which involves the removal of the nucleus from an oocyte (an immature egg cell) and its substitution (via injection) by a single nucleus taken from a somatic cell. With a few chemical tweaks, the new cell begins dividing like a fertilized egg. In the case of Dolly, the donor nucleus was taken from a sheep mammary cell. The arrival of “Dolly” transformed contemporary views of the stability of the genome in somatic cells since it has been previously concluded that a complete resetting of the genetic program in any somatic cell was not possible.
In 2002, Colman joined ES Cell International, a Singapore-based company that specializes in developing human ES cell lines. Five years later, he moved to A*STAR to take advantage of iPS cells to look into aging mechanisms. He has abandoned nuclear-transfer technology and now uses ES cells as experimental controls only.
Currently, most researchers at Colman’s laboratory are devoted to developing iPS cells to model several human genetic diseases including Hutchinson–Gilford progeria syndrome, a premature aging disease. The disease is caused by a single mutation of the ‘nuclear lamin A’ gene. The mutation leads to the production of a mutant protein called progerin, which affects the integrity of nuclear envelope and triggers the acceleration of aging. Colman explains that while this mutant protein is produced in most cells, only certain cells suffer and accelerate aging. “We are trying to understand why, in this disease, only certain cells are affected. A patient’s central nervous system remains fine, but they develop cardiovascular problems.”
His team’s approach is to take fibroblast skin cells from a patient and reprogram them using retroviruses carrying four transcription factors. This results in the production of many iPS cells, which can then be used to study the pathology of the disease in the early stages of development. Colman says that aspects of this particular disease resemble normal aging, so “studying progeria could lead to a better understanding of normal human aging.”
Comparison of 2D colony on surfaces (left) with 3D cultures on cylindrical microcarriers (right) for the growth of human ES cells. 3D-cultured cells, developed by Steve Oh, develop to 2–4 times higher densities than 2D colonies.
New technologies
Although iPS cells have taken up much of the limelight over the past few years, the majority of papers published by A*STAR researchers still involve the use of ES cells. Many scientists working on both types of cells share a common challenge: how to make a variety of desired cell types efficiently. Researchers have been struggling to produce a sufficient number of usable cells for possible therapeutic applications. An equally important issue is the safety of the stem cells, as past research has shown that ES and iPS cells can switch spontaneously into cancer cells.
Steve Oh and Andre Choo at the A*STAR Bioprocessing Technology Institute are striving to address solutions to some of these problems1,2. Oh, a principal scientist in the Stem Cell Group, has invented a method to attach human ES cells to rod-shaped, cellulose microcarriers and grow them as a three-dimensional cluster in suspension cultures. The conventional method is to culture human ES cells on two-dimensional surfaces in a Petri dish, but this approach has limitations in terms of the yield of cells. Oh’s approach allows long-term, serial growth in suspension cultures and achieves 2–4 times higher cell densities than using conventional cultures.
For their next step, Oh and his colleagues have triggered the aggregated cells to differentiate into cardiomyocytes (heart muscle cells) in serum-free culture conditions. But the major problem is that a small number of stem cells in these clusters of mature cells remain undifferentiated, which can develop into tumors known as teratomas. Choo, senior scientist in the Stem Cell Group, has developed several antibodies that bind selectively to undifferentiated ES cells and kill them. “Then we can collect pure populations of cardiomyocytes,” says Choo. He is aiming to improve the accuracy of the method to make it possible to guide antibodies to the remaining undifferentiated populations of cells. “We could then target cancer stem cells, or another disease indication in the future,” Choo adds.
These techniques are promising but there are still a number of problems to be solved. For example, Oh says ES cells tend to differentiate spontaneously in suspension cultures, so he needs to develop another method to prevent this from happening. ”Transferring the cells to larger volumes of culture is a major challenge,” he says. Work is ongoing to scale the process to controlled bioreactors of several litres.
An immunofluorescence image of an oocyte. Davor Solter has been studying the myth of the oocyte-to-embryo transition, and takes a cautionary stance on human ES cells and human iPS cells.
Back to basics
Despite the rapid progress in stem-cell research over the past few years, Solter has taken a step back from stem cells to examine how an oocyte transforms into an embryo in such a short period of time. “Development starts not from fertilization but goes way back to the processes involved in making oocytes. Sperm cells only contribute DNA and maybe some other things,” he says. His laboratory, which he runs in collaboration with Barbara Knowles, a professor and principal investigator, is focusing on determining the molecules and processes that are involved in the activation of embryonic genomes in oocytes.
Although there are some cases of treatment using adult stem cells, such as for serious burns, Solter raises serious questions regarding the potential therapeutic application of ES cells. “For example, if pancreatic stem cells are applied by injection into blood vessels, they may not know what to do. How that is going to work is totally unclear,” he says. He also has doubts about the eventual clinical use of iPS cells, suggesting instead that iPS cells could be bypassed with yet another technology. Solter says researchers may soon be able to differentiate fibroblasts directly into organs or neurons, in which case iPS cell technology would become another transient development in the saga of stem-cell therapy.
Not everybody agrees with that opinion, however. “I personally don’t think direct trans-differentiation will be easier in general,” argues Colman. “That approach will be difficult to use if large numbers of cells are needed, since often the differentiated cells cannot divide well. It may remain preferable to grow up large numbers of iPS cells and then differentiate these into large numbers of the desired cell type.” Colman says convincing other scientists that their cell types behave in the same manner as cells in patients is as difficult a challenge as making a variety of adult-like differentiated cell types.
Beyond all the debate in stem-cell research, researchers universally agree that a better understanding of the underlying developmental biology is essential and could lead to remarkable and exciting outcomes in the future. “At the present time, we do not know how to exactly apply differentiated derivatives of ES cells for cell and tissue replacement,” Solter says. “In many cases these cells would have to precisely integrate in the right place in the right organ and it is not clear how to achieve that.”
