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An Interview with Dan Kaufman

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A note from his month’s Feature Principal Investigator, Dan Kaufman. Dr. Kaufman is currently an Associate Professor in the Department of Medicine and Associate Director of the Stem Cell Institute at the University of Minnesota. His time is divided approximately 80% research and 20% clinical work in their Blood and Marrow Transplant Program.

‘I have been fortunate to have a series of outstanding mentors and work environments throughout my research career. I first began doing immunology research as an undergraduate student at Stanford University where I began in the lab of Dr. Garry Fathman. I worked most closely with Dr. Judy Shizuru who was a graduate student and then a post doctoral fellow in the Fathman lab. This research on use of monoclonal antibodies to deplete specific T-cell subsets as a way to improve pancreatic islet transplantation in mouse allograft and xenograft models was a wonderful introduction to translational immunology research. These studies led me to apply to combined MD/PhD programs and I wound up doing this in my home state (Minnesota) at the Mayo Clinic. Here I continued in immunology research but shifted focus to work on human natural killer cell (NK) cell biology in the lab of Dr. Paul Leibson. Overall, this again was a great research experience where I was able to identify signaling pathways that inhibit human NK cells when interacting with HLA class I molecules. This was at a time before killer immunoglobulin receptors (KIRs) or most other receptors on NK cells had been identified, but we were able to make great progress using the human NK cell clones that were the basis of studies in the Leibson lab. Following this I went to the University of Wisconsin-Madison to pursue residency in Internal Medicine. I knew I was interested in doing more research as well as pursuing clinical training in hematology and blood and marrow transplantation (BMT). I was fortunate to be in the right place at the right time when Dr. James (Jamie) Thomson published the first studies on human embryonic stem cells (hESCs). Soon after that publication I contacted Jamie to explore the possibility to join his research group to study hematopoiesis from hESCs. I actually had a break in my clinical training in early 1999 and was able to spend most of the next two years in his lab as a post-doctoral fellow. During this time I was able to establish the initial system to produce different human blood cell populations from hESCs. We also did studies using nonhuman primate (Rhesus monkey) embryonic stem cells which I primarily used to study endothelial cell development with the assistance of Dr. Bob Auerbach who is a pioneer in endothelial cell biology and angiogenesis studies. Following this fellowship training I moved back to Minnesota to take a faculty position at the University of Minnesota in 2002.’

 

 


An Interview with Dan Kaufman

 

By Carla Mellough

What was your original motivation for becoming a researcher in the field of stem cells? How has this motivation evolved?

My motivation for becoming a researcher in the stem cell field again really developed from my being at the University of Wisconsin when hESCs were first derived. It was easy to see that these provided an excellent system to study human hematopoiesis rather than having to rely on murine systems, the mainstay of this area of research. Additionally, we could start with a cell population that was a precursor to hematopoietic stem cells (HSCs) rather than just isolating human HSCs from cord blood, bone marrow, or peripheral blood. Furthermore, hESCs provided a novel opportunity to develop new stem cell therapies from this pluripotent cell population. Perhaps the most important thing at the time (and is still much the case) is that the field of human stem cell biology was very wide open with many interesting directions to pursue both in terms of basic biology as well as therapeutic applications.

Having been working with human pluripotent stem cells now for over eleven years I have been fortunate to see this field grow enormously. However, my motivation and interest with working on hESCs and now human induced pluripotent stem cells (iPSCs) is much the same. There is still the need to use these systems to understand basic human hematopoiesis. Additionally, clinical translation of these cells, especially towards hematopoietic therapies remains slow but gaining ground. While the numbers of people that have entered this field over this past decade is incredible, it is still somewhat surprising that many of these basic questions and opportunities still remain unaddressed.

 

Much of your work is focused on the study of hematopoietic and endothelial development. Your recent article in Blood demonstrates that natural killer cells (NKs) derived from hESC are more effective in clearing diverse tumor cells than NKs derived from the umbilical cord and thus may serve as a source of antitumor immunity therapy. In your experience, what appear to be the main differences between hESC-derived and umbilical cord-derived NKs which mediate this effect?

One of the first research directions I pursued in my independent lab at the University of Minnesota was to derive lymphocytes from hESCs, as no one had done this up to that point. I chose to pursue NK cell development for two reasons. One being my familiarity with these cells as mediators of innate immunity based on my graduate studies. Second, Dr. Jeff Miller at the University of Minnesota had developed a culture system using stromal cells and defined cytokines that was able to efficiently produce NK cells from CD34+ cells isolated from human umbilical cord blood (UCB). Therefore, we rather simply just compared use of the UCB CD34+ cells to our hESC CD34+ cells in this culture system. We found that indeed we could routinely produce NK cells using this system. Somewhat surprisingly, when we got better at producing our hESC-derived NK cells and then tested them in an in vivo model of antitumor activity, we went on to demonstrate that the hESC-derived NK cells had more potent antitumor activity than the UCB-derived NK cells (see Figure). We found that this was due to the hESC-derived NK cells being a more homogeneous population of CD94+/CD117low/- cells that have potent cytolytic activity. The UCB-derived cells (as Drs. Miller and Verneris at the University of Minnesota had previously shown) were a mixture of CD94+/CD117- and CD117+/CD94- cells. However, only the CD94+ cells have cytolytic activity. We have more recently been able to derive more similar NK cells for iPSCs and find similar production CD94+/CD117- homogenous NK cells. Additionally, we have now been using NK cells isolated from peripheral blood (PB) as a more suitable control cell population and find that the ES- and iPSC-derived NK cells are very similar phenotypically and functionally to peripheral blood NK cells.

 

How quickly do you think this could be transferred to the clinic?

This research on NK cell development from human pluripotent stem cells closely corresponds to clinical work in the BMT Program at the University of Minnesota that is testing adoptive immunotherapy using NK cells (isolated from peripheral blood) to treat cases of chemotherapy refractory cancer. Dr. Miller has led these trials and we have had many cases of acute myelogenous leukemia (AML) put into remission using NK cell-based therapies. In some cases this can be followed up with a standard allogeneic BMT to provide long term remission or cure of these AML cases. Based on this intriguing track record, I am eager to pursue use of hESC and/or iPSC-derived NK cells that may also be suitable for clinical therapies against refractory cancer, or possibly lethal/chronic infectious disease such as HIV/AIDS. The main hurdles to this hESC and/or iPSC-derived therapies are 1: producing our hESC/iPSC-derived cells in a GMP compliant manner and 2: scaling up our process to generate enough cells suitable for human therapies (rather than just treating mice). We have made great strides towards both of these goals. However, the time that it will take for this to be transferred to clinic is clearly directly related to the funding available. Having just one or two graduate students or postdoctoral fellows working on this project obviously leads to slower progress than having a more numerous dedicated team. To achieve this goal, we are currently pursuing a number of funding opportunities such as from the NIH, private foundations, or possibly other sources.

 

What do you feel is the most challenging aspect of your job?

The most challenging aspect of my job is clearly funding of the research that we wish to pursue. There are so many intriguing directions and opportunities. However, funding levels continue to drop. We are not only pursuing our studies on hematopoiesis and NK cell development but also have projects related to developing vascular cells (endothelial and smooth muscle cells) suitable for treating myocardial or peripheral ischemia. We also have a project on generating osteogenic cells from hESCs and iPSCs that are suitable for fracture repair. These are all mesoderm-derived cell lineages and actually closely developmentally related. However, with limited grant support we may need to cut back on some of these research directions. While it has always been considered that “good research will always find a way to get funded,” this may not really be the case now. We all know instances of superb researchers and important research projects being curtailed due to lack of funding. I think this problem will only continue to grow as the NIH funding level drops and other resources may become more scarce. Of course, some states have provided other opportunities for funding, though unfortunately this is not the case in Minnesota where there is no dedicated specific funding for stem cell research. Predictably, this lack of necessary resources hampers both basic science advances as well as clinical translation of stem cell based therapies.

 

There has been a huge shift in public thinking about the use of stem cells for research and to ameliorate human disease. In your opinion, what are the main barriers that still remain for the clinical translation of hESC? How do you foresee these being overcome?

I am not so sure there has been a significant shift in attitudes. After being in the stem cell field for over ten years, I can still see that there remains considerable confusion by the general public over the potential of different stem cell populations to treat human diseases where there are not currently effective therapies. For example, there is still confusion about the source and developmental potential of adult (or tissue specific stem cells) compared to pluripotent stem cells. Sham therapies purported to use stem cells are widely advertised on the internet. Without proper knowledge and properly conducted clinical trials, these practices lead to dangers for patients, rather than benefits. Also, the debate and some of the unfortunate negative connotations regarding hESCs has still not subsided. Indeed, many people and groups who have opposed hESC research and therapies continue to suggest that advances made with adult stem cells as well as iPSCs means that hESC-based research is no longer necessary. This is clearly not the case, as the strong consensus in the field is that hESCs still provide a “gold standard” for pluripotent stem cell research. Indeed, as we see iPSCs have more diversity in their developmental potential and identifying which putative iPSC lines are truly pluripotent remains a challenge. Therefore hESC-based studies remain as necessary as ever, if not even more so.

The recent legal ruling in the USA that may effectively shut down NIH support of hESC research further emphasizes the continued problems in the hESC research field. Hopefully, this issue will be solved soon. However, even this (potentially) temporary block in NIH support leaves a large impact on the hESC field and may inhibit young or new investigators from pursuing this critical and exciting area of research.

It is certainly gratifying to see hESC-based therapy moving into clinical trials. A protocol using hESC-derived oligodendrocytes was approved by the FDA in January 2009, though those trials are still on hold due to safety reasons. A well-conducted clinical trial with hESC-derived cells as planned with this therapy (or therapy from other suitable cell populations) will be a great boost to the stem cell field. This is especially true for those interested in moving other hESC- and iPSC-derived cells towards additional novel clinical trials, such as for diabetes, vascular repair, or cancer therapy. Indeed, one of the other most intriguing areas is development of retinal pigmented epithelial cells such as work done by Dr. Peter Coffey in England, as described in a previous issue of this Stem Cells Portal.

As alluded to, our ability to now move hESC and iPSC based therapies towards the clinic is a key next step in the field. There remain a number of issues, including expanding the availability of hESCs and potentially iPSCs that meet current good manufacturing practices (cGMP) criteria. Such cell lines have now been produced by multiple groups, though access and testing of these cells may still be limited. There is also the need to scale up the number of cells for effective therapy in humans, as is necessary in the case of our hESC-derived natural killer cells. While we can now easily make several million cells in culture which are enough to treat a mouse, we still need to invest in the development of methods that will allow us to routinely produce the 108 cells necessary for human clinical trials. However, I believe this is a very achievable goal, and perhaps more realistic than producing 1012 red blood cells that are necessary for a unit of transfused red blood cells. Clearly it is an advantage for some therapies such as for retinal cells or treatment of spinal cord injury where only 105 -106 cells may be needed for each patient.

Another barrier will be effective immunosuppression, as hESC-derived cells and potentially iPSC-derived cells will be allogeneic. However, the clinical transplantation field has been doing allogeneic transplants for decades and immunosuppressive medications such as cyclosporine, tacrolimus, or mycophenolate may all be effectively utilized for these new stem cell based therapies. Obviously, clinical protocols will need to be designed to offer optimal immunosuppression to prevent graft rejection without unwanted toxicities.

 

In your opinion, what do you consider to be the most important advance in stem cell research over the past 5 years?

It is clear that the most important advance in stem cell research over the past five years is the development of iPSCs. While there was some previous evidence based on cell-fusion and related studies that this method of cellular reprogramming might be feasible, it was not considered seriously until the pioneering studies of Yamanaka which demonstrated that cellular reprogramming to a truly pluripotent state using just a few defined genes was possible. The most remarkable aspect of iPS cells is the routine applicability of this method that has now been utilized by hundreds of investigators worldwide. This reproducibility and advances in reprogramming technology is in contrast to many studies done with adult stem cells that suggested that they might have multi-potent or pluripotent capabilities. While there have been reports of adult stem cell plasticity by many labs worldwide, the methods used and the cell population studied are typically quite heterogeneous. This has made the work on adult stem cell plasticity difficult to reproduce between groups. In contrast, iPS cells have been produced by so many groups that its acceptance and advances are growing at a continuing accelerating rate.

Currently, a key issue with iPS cells is to achieve lineage-specific differentiation to therapeutic cell populations of choice. In this way the iPS cell field really has the same challenges that those of us working with hESCs have been facing for the past decade. Indeed, in my area of hematopoiesis research, many groups have been trying to isolate HSCs with the ability to provide long-term multi-lineage engraftment when transplanted into in vivo models, typically immunodeficient mice. Despite many attempts to isolate hESC-derived HSCs, robust in vivo engraftment has still not been achieved. Indeed, studies with mouse ESC that preceded hESCs by over a decade are also not able to mediate efficient engraftment without genetic modification of the ES cells. Now those coming into the field who wish to produce hematopoietic cells from iPSCs (especially iPSC-derived HSCs) are facing the same hurdles. We are currently trying to advance new models to both identify definitive hESCs with engraftment potential as well as utilize novel transplantation systems that might provide key host factors necessary for engraftment.

There are a couple of recent studies that suggest lineage specific differentiation of iPSCs is less efficient than hESCs. This has been shown for studies of neural development, even using an iPS cell line without integrating viruses. It is also likely that iPSCs retain “epigenetic memory” or other mechanisms that facilitate improved derivation to some cell lineages over others. Indeed, recent data by Dr. George Daley’s group demonstrates the role of epigenetics on the developmental potential of different iPSC lines. Typically, producing the lineage from which the iPSC was originally derived is easier than producing other cell populations. For example, reprogramming a cord blood cell to an iPSC line that can more efficiently differentiate into hematopoietic lineages as compared to an iPSC derived from a dermal fibroblast.

Based on publications and work presented at the recent ISSCR meeting, it is clear that direct reprogramming is the “next big thing” in stem cell biology. While only a couple of studies have been published to demonstrate direct reprogramming for pancreatic and neural development in murine systems using a few defined genes, considerably more advances are likely in the next few years. However, it will be necessary to have appropriate reporter systems built into human cells to enable direct reprogramming in a human system. Also, there is considerable interest in converting these reprogramming systems to use drugs and small molecules rather than genes. While this would likely lead to more direct clinical applications, challenges certainly remain.

 

What are your hopes for the future stem cell research and clinical translation in your specialist area?

As a physician/scientist, the stem cell field is an ideal area. There remain so many key interesting and important basic research questions that remain to be answered, as well as the opportunity to move pluripotent stem cells into novel clinical therapies that the possibility and potential of these cells is essentially endless. Clearly these two aspects are intertwined, as improved understanding of basic stem cell biology will improve our ability to derive therapeutic cell populations of choice. It is also interesting to see this field moving in new directions. For example, stem cells were originally touted to treat diseases such as Parkinson’s disease, diabetes, or cardiovascular injury where the missing or damaged cell population is quite well defined. However our studies on derivation of lymphocytes, especially NK cells, now leads to use of hESCs and iPSCs to treat lethal diseases such as cancer or even HIV/AIDS in a new and more fully effective manner. Obviously more research and investment is needed but I think over the next few years real advances in bringing these types of new stem cell-based therapies to the clinic will be a reality.

 

What do you think are the rewards and need for training in stem cell biology?

While there are many challenges in this field, it should be emphasized that there are at least as many rewards. In addition to the scientific and potential clinical advances, all investigators who work at academic institutions clearly know the personal reward to train new investigators in this field. As the area of stem cell biology and clinical translation continues to expand, clearly more specific training in this area is needed more than ever. In my lab I have had the pleasure to train new investigators at all levels including a high school student, undergraduates, graduate students, postdoctoral fellows and clinical trainees. I have also participated in many training courses. Perhaps most importantly I am principle investigator on a new NIH sponsored T32 training grant in “Stem Cell Biology”. This training grant was recently funded this fall and will support graduate student trainees working with stem cell investigators at the University of Minnesota. Surprisingly, the NIH supports relatively few training programs in Stem Cell Biology and/or Regenerative Medicine. Indeed, even finding an institute at the NIH interested in supporting this type of training was difficult.

In addition to this T32 training grant the University of Minnesota now has a Masters Degree program in Stem Cell Biology. This approximately 18 month program allows both didactic and lab-based training in this field. Additionally, graduate students in many programs at the University of Minnesota have the option to get a “minor” in Stem Cell Biology. Eventually I would hope to develop a full fledged PhD graduate program and a full academic Department of Regenerative Medicine. I think being able to tie in all aspects important to push this field forward both scientifically and clinically will benefit by being more coordinated in this fashion. While a few institutions have developed such a department, these remain few and far between. However, encompassing aspects including stem cell biology, immunology, tissue engineering/bioengineering and clinical training is vital to promote the most efficient future growth and advancement in this area. Additionally, as both small biotech companies and larger pharmaceutical companies are pursuing different aspects of stem cell based research and clinical applications, more investigators trained in this field will be needed at all levels.

 

Which important basic biology questions remain to be answered, in your opinion?

An example of one of the aspects of the hESC/iPSC field that needs to be understood is to identify how the cellular signals and mechanisms that are essential to maintain cell pluripotency also affects the differentiation capacity of these cell populations. Both our lab and many other groups that I have discussed this with clearly see that hESCs, and likely iPSCs, have differing ability to produce a cell lineage of choice depending on how they are maintained as undifferentiated cells. For example, we routinely maintain our ES cells on MEF feeders using serum-free media, and this is quite suitable for subsequent stromal based differentiation into hemato/endothelial cells. However, trying to move these cells into a “spin EB” system as developed by Elefanty’s group does not lead to successful EB generation. However, adapting the hESCs using trypsin and lower density MEFs does then lead to efficient “spin EB” mediated differentiation that works quite nicely in our hands. Similar differences have been found for development of cardiac and other lineage cells. Additionally, use of completely defined media such as is commercially available seems to be quite good at maintaining undifferentiated ES cells. However, these cells can be more difficult to subsequently differentiate into defined cell populations. This issue emphasizes the dual need to continue to evolve or improve methods for lineage specific differentiation; however, at the same time investigators need to “stick with what works” to most definitively test the characteristics of a differentiated population of choice. Again, many investigators who are new to this field are finding out about these issues that are not typically published. Important collaborations such as through the International Stem Cell Initiative led by Dr. Peter Andrews (University of Sheffield) are starting to now coordinate groups interested in different germ cell layers or cell populations to try to standardize and/or catalog methods utilized for both hESC/iPSC culture as well as differentiation. This will be a complex endeavor as there are many potential methodologies or factions that must be considered. However, this aspect of pluripotent stem cell biology is a critically important future direction.

 

kaufmanfigure

FIGURE: hESC-derived NK cells demonstrate clearance of established tumors xenografted in mice with higher efficacy compared with NK cells generated from UCB. NOD/SCID mice were inoculated with luc+ K562 tumor cells, and cells were allowed to engraft for 3 days before animals were given 1 systemic (intravenous) infusion of NK cells. All hESC- and UCB-derived NK cells were injected after 30 to 35 days of culture. Mice were monitored by bioluminescent imaging at days 0, 4, 7, 14, and 21. In addition, some mice demonstrating tumor regression were monitored long-term (up to 8 weeks) for tumor recurrence. This figure shows representative in vivo bioluminescent images of animals at the day of tumor inoculation and 21 days after tumor inoculation. Mice treated with UCB-derived NK (UCB-NK) cells typically display slower tumor progression. All mice treated with hESC-derived NK (hESC-NK) cells displayed a complete clearance of tumor cells.

 

More information about the Kaufman research team can be obtained from the lab website and a video on the NK cell work can be viewed on YouTube. For more information on the hurdles facing bringing pluripotent cell based therapies to the clinic please see Dan’s extensive review in Blood.

 

Kaufman Lab's Featured Paper