Celebrating 10 Years of hESC Lines: An Interview with Ariff Bongso

About Dr. Bongso

Professor Bongso received a Doctor of Veterinary Medicine in 1970 from the University of Ceylon in Sri Lanka. He then earned a Commonwealth scholarship to pursue postgraduate studies at the Ontario Veterinary College of the University of Guelph in Canada. Here, he obtained an M.Sc. in 1973 and a Ph.D. (with distinction) in 1976 from the Department of Biomedical Sciences, focusing his research on amniocentesis and the sexing of embryos using animal models. In 1987, he was invited to join the laboratory of Professor S. S. Ratnam, a pioneer of in vitro fertilization (IVF), in the Department of Obstetrics and Gynecology at the National University of Singapore. Here, his research focused on IVF techniques and human embryonic stem cells (hESCs). In 1990 Professor Bongso's group identified several factors released by human fallopian tubes that have a major role in fertilization and early embryonic growth. These findings were used to develop the culture medium that is used at almost all IVF clinics today. In 1994 Professor Bongso's group isolated the first ESCs from blastocysts, an important step in the development of hESC lines by Dr. James Thomson 4 years later. In 2000 Dr. Bongso co-founded a company called ES Cell International (ESI), which focuses on cardiovascular disease and diabetes. In 2002 his team demonstrated that human fetal and adult fibroblast feeders can be used in place of mouse feeders to support undifferentiated growth of hESCs, addressing one major safety concern for the clinical use of hESCs. He is currently Research Professor and was Scientific Director of the Assisted Reproductive Technology program in the Department of Obstetrics and Gynecology of the National University of Singapore. He is also currently Co-Leader of the Regenerative Medicine Themed Group of the Yong Loo Lin School of Medicine, National University of Singapore. His research examines the elimination of teratoma formation by hESCs, using hESCs for pharmacological screening assays, the differentiation of hESCs into germ cells, the derivation and application of Wharton's jelly stem cells (WJSCs), and the interaction of hESCs and WJSCs with nanofibrous materials. His numerous awards include the 2005 ASEAN Outstanding Scientist award, the 2003 Excellence for Singapore Award, the 2002 National Science Award, Singapore, the 2002 Asian Innovation Award (Gold), and the 1997 Outstanding University Researcher Award: National University of Singapore. Professor Bongso is a Fellow of the Sri Lanka College of Obstetricians & Gynaecologists and a Fellow of the Royal College of Obstetricians & Gynaecologists, United Kingdom.

“Having a Strong Background in Comparative Mammalian Embryology Has Been an Asset”

Dr. Bongso has taken an interesting path to stem cell research, beginning in veterinary medicine, and continuing on to comparative mammalian biology.

“During my undergraduate days, I was offered a place in medical school which I accepted and left after 3 months to take up an undergraduate program in Veterinary Medicine. This was against my parents' wishes. I did not think at that time that I could take the responsibility of someone else's life into my hands. Perhaps I was too emotional. Today when I look back I am delighted that I completed a 5-year program in Veterinary Medicine and then moved onto a Ph.D. program to follow a research career in biomedical sciences where I could translate my ideas from animal to human. Having a strong background in comparative mammalian embryology has been an asset. I had a strong passion for reproductive biology in companion, agricultural, and wild animals as their reproductive physiology was so different and the diversity of the animal kingdom including the human excited me. The sudden shift from human medicine to veterinary medicine to me was a defining moment in my education.”

He began his research in Singapore studying the problems associated with human IVF. At that time, many in the scientific community were puzzled as to why human IVF pregnancy rates worldwide were only 15%–20% when in domestic animals the rate was more than 60%. “The reason for the difference was very obvious to me because in domestic animals in vitro fertilized embryos were transferred to the uterus on day 5 at the blastocyst stage, while in the human, embryos were being transferred to the uterus on day 2 at the four-cell stage. In vitro conditions to grow human embryos at that time were suboptimal, and embryos could not be grown beyond day 2 to the blastocyst stage. Also, in the human there was an ‘in vitro embryonic block’ between day 2 and day 3 causing arrest of embryos if culture conditions were suboptimal. This was a crucial stage of embryonic development because transcription was being handed over from the egg to the embryo and in vitro conditions had to be more fastidious to help bypass the block. Domestic animal embryos were not as ‘fussy,’ and their in vitro blocks were at different times. To overcome the in vitro embryonic block and grow embryos to the blastocyst stage so as to boost pregnancy rates for human IVF, in 1988 I decided to develop a more sophisticated in vitro system to grow human embryos to day 5. My team and I stripped the inner epithelial lining of the ampullary region of the human fallopian tube and grew this lining as a bed of cells. We also collected and analyzed the composition of small volumes of fallopian tubal fluid and developed our own culture medium based on the formulation of this tubal fluid. This medium was used to bathe the bed of tubal epithelial feeder cells on which we grew day 2 embryos, thus mimicking the physiological conditions of the fertile fallopian tube. We were pleasantly surprised to find that nearly 80% of day 2 embryos grew to the blastocyst stage, and the IVF pregnancy rate doubled to 42%.”

“The Stage Was Set for Human Embryonic Stem Cell Biology”

“This development I believe gave us an advantage and led us to isolating stem cells from blastocysts because we were one of the few IVF centers worldwide growing blastocysts. We were able to recognize all kinds of blastocysts (early, expanding, fully expanded) with inner cell masses of various sizes and then developed a staging system to grade blastocysts so that one could select the best blastocyst with the highest potential of pregnancy before transfer. My background of comparative embryology taught me that the inner cell mass was responsible for producing the three primordial germ layers from which all the tissues of the human body developed. It was at this point in 1994 that I got interested in trying to separate the inner cell mass cells, expand them in culture without differentiation, and then attempt to direct them along a desirable lineage to prepare tissues to cure incurable diseases via transplantation therapy. It was perhaps a natural progression. Our first successful attempt at isolating stem cells from human embryos was in November 1993 when we presented our findings at the World Embryo Transfer Society annual meeting in Melbourne, Australia. More detailed descriptions of our work were later published in November 1994 in the journal Human Reproduction.” Interestingly, Dr. Bongso's laboratory did not continue this work to attempt to produce stem cell lines. “This was a once and for all experiment to find out whether one could isolate stem cells from human embryos. It was not meant to be continuous to generate cell lines as my passion at that time was still human IVF. I may have pursued the development of a hESC line further if our published work drew more enthusiasm. This perhaps may have been because it was published in a Reproduction journal or perhaps because the potential of these mysterious cells were unnoticed at that time, although we explicitly explained in our paper the tremendous potential these cells had for future transplantation therapy.”

Scientists Should Now “Concentrate on the Translational Aspects to Help Us Make hESC-Derived Tissue Therapy a Reality”

Today, Dr. Bongso believes that it is most important to focus on making stem cell therapy a reality. “The future directions of the research, I believe, must be translational where there is a final benefit to mankind. The journey is a long one but I believe that with a lot of commitment and hard work it will be a fulfilling one. To me, the aspects of hESC research that require urgent attention are (a) eliminating the concern of potential teratoma formation, (b) eliminating potential immunorejection, and (c) developing methods to scale-up hESC numbers for clinical application. Many scientists have already shown successful engraftment of hESC-derived tissues in animal models for a multitude of diseases, but their work cannot be taken to the clinic until the above three issues are resolved. It also would be useful if some of the preclinical validation carried out on laboratory animals is extended to nonhuman primates to give more confidence to our patients that hESC-derived transplantation therapy can be applied to the human.

The biggest technical obstacle to overcome is the issue of potential teratoma formation induced by rogue undifferentiated hESCs residing in the hESC-derived cell population. Any new protocol to overcome this problem must be foolproof. This is even more important than personalizing tissues to prevent immunorejection. We cannot commit to our patients that stem cell-based tissue therapy is safe until this is resolved. One case of tumorigenesis in a patient after hESC-derived transplantation therapy is enough to set back the tremendous therapeutic potential these cells can offer.”

“The Induced Pluripotent Stem Cell Approach Is Very Elegant and Novel … but May Be Limited to Being a Useful Model to Study Human Disease”

“I strongly believe that hESC research will be impeded in countries where such research is not accommodated and the use of embryos is not allowed for creation of more hESC lines. I believe that the quickest way of taking hESC research to the clinic is by the development of several repositories of hESC lines where panels of such cell lines can be tissue matched to patients to eliminate the issue of immunorejection. There are millions of surplus embryos frozen in tanks all over the world, and if these are to be discarded they will be useful material to meet the objective of tissue matching if informed patient consent and institutional review board approval can be sought. It is time that several repositories of hESC lines of various ethnic backgrounds be developed with a global coordinated effort between countries in which hESC research is not controversial. The discovery of the induced pluripotent stem (iPS) cell approach is very elegant and novel and has helped to bypass the ethically sensitive issues of the use of spare IVF embryos for hESC derivation. However, iPS cells are still pluripotent and induce teratomas, and I believe that it may therefore be limited to being a useful model to study human disease and not for clinical application because of this issue. Also, I would presume that total reprogramming would be necessary for normal epigenesis, and this may not be provided fully by using a small package of pluripotent genes. The difference with embryo-derived hESCs is that epigenesis may be much more normal and complete. I am a proponent of Mother Nature and established paradigms of embryonic development. My message to the next generation of scientists in stem cell research is to move slightly away from the basic aspects of stem cell research as we have had a long haul at this. They should build on work already done and concentrate on the translational aspects to help us make hESC-derived tissue therapy a reality.”

“Success Never Comes Without Hard Work”

My advice to young scientists is that if you are married, you need a very supporting and understanding spouse because as a budding scientist you have to spend a lot of time scanning and reading scientific literature, carrying out your experiments for your research even if it has to run into the nights, analyzing data, and documenting your work. Stem cell research is very demanding as the field is very competitive and labor-intensive. A reporter once wrote in Asiaweek that I was toiling in obscurity working hard on these mysterious cells being unnoticed by the wider scientific community. My motto is that success never comes without hard work. Many of the diseases plaguing mankind are on the rise, and doctors don't seem to have full control over the devastating ones. This is sad. I believe that the greater part of the research we do should benefit mankind in the shortest possible time. I am confident that the great minds of many stem cell scientists worldwide working in a focused, synergized mode will bring hope to many of those with incurable illnesses. This hope guides me in my daily life. 1

Figure 1. Ariff Bongso, Ph.D. National University of Singapore.