Jeanne Loring interview: optimism on clinical translation of IPS cells

One of my favorite stem cell scientists is Jeanne Loring of Scripps. She does great science and when you ask her questions, she frankly states her opinions and is clearly a gifted educator at heart too.

Below is a Q&A interview I did with Jeanne on key issues of clinical translation of iPS cells. You will see that Jeanne is very optimistic about the eventual clinical use of iPS cell-based medicines.

My Qs are bolded and her answers are italicized. Enjoy this Q&A.

1. While there is some disagreement over the exact number, it seems that  on average, each iPSC line has a very small number of mutations. Some  mutations are present in the somatic parental cells of origin of the  iPSCs, while others may occur rarely during the iPSC production process. What is your feeling for the functional significance, if any, of these genetic changes?

Jeanne: I think this is an attractive idea that turns out to just not to be true.  There were several reports that reprogramming causes mutations, but the studies were flawed because they didn’t consider the existing heterogeneity of the fibroblasts used for reprogramming.  The paper from Flora Vaccarino’s group at Yale has solved this issue, I think (Abyzov, et al, 2012. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012 492:438-42).

We are all mosaics of cells with slightly different sequences, and the mosaicism shifts in dividing cells as they acquire sequence changes (note that I did not call these “mutations”). Since healthy people have this mosaicism, we assume that it is not harmful.

Another issue is the sequencing technology itself- there is an intrinsic error rate, and to confirm single base changes we need very deep sequencing and many replicate samples.  Most stem cell researchers are not also expert in genomics, so it’s easy to be misled.

2. iPSCs also exhibit epigenetic differences from hESCs. Is it known what if anything these differences mean for the biological behavior of the iPSCs? Given that the epigenome is inherently dynamic, is there concern that iPSCs could “drift” during preparation?

Jeanne: If you were asked, for a bet, to distinguish hESCs from iPSCs by their epigenomes, with one exception, I’m afraid you would lose.  The exception is the X chromosomes in female cells.  We females have two X chromosomes, while males have only one, so to balance the activity of the genome, we turn one of the two X’s off. When iPSCs are first generated from female cells, one of the X chromosomes remains inactive. About half the time, the inactive X in iPSCs becomes active over time in culture, becoming indistinguishable from female hESCs, which start out with two active X’s.  If you were given a set of male iPSCs and hESCs, and asked to distinguish them, you’d be completely out of luck. There are no other consistent differences between iPSCs and hESCs.

I should have asked you to bet on this before telling you the secret…perhaps I’ve missed my chance to win.  Actually, this is not a secret; those who are brave enough to tackle our admittedly dense opus on DNA methylation already know the answer (Nazor et al, “Recurrent variations in DNA methylation in human pluripotent stem cells and their differentiated derivatives”. Cell Stem Cell 10: 620-634. (2012)); it’s a tough read, so I think that most people are waiting for it to come out on video…

The approach is summarized in this quote from the paper:  “To obtain a comprehensive view of hPSC-specific epigenomic patterns, we collected 136 hESC and 69 hiPSC samples representing more than 100 cell lines for analysis. In order to establish expected variation in human tissues, we collected 80 high-quality and well-replicated samples representing 17 distinct tissue types from multiple individuals. Finally, we selected 50 additional samples from primary cell lines of diverse origin to control for any aberrations that may arise as a general, non-hPSC-specific, consequence of in vitro manipulation”.

Translation:  we looked at a LOT of cell lines and samples, not just a few.  Shinya Yamanaka points out in a perspective in the June 2012 issue of Cell Stem Cell that the consistent story from large studies is that iPSCs and hESCs are the same; from small studies, that they are different. If you look closely at a small number of cell lines, you are bound to find differences, between iPSC and hESC, as well as among hESC lines and among iPSC lines.  If you look at a lot of cell lines, those differences turn out to be insignificant.

3. In addition, it seems that iPSCs can possess epigenetic memories of their cell of origin. For example, an iPSC made from blood cells has been reported to be more adept at differentiating into blood cells versus an iPSC made from skin, which is better at making skin. Do these memories impact the therapeutic potential of iPSCs?

Jeanne: This is another attractive idea that doesn’t hold up well to examination.  I wonder how many people have actually tried this?  We (and others) have found that these “memories’ are inconsequential and transient.  The epigenome of iPSCs continues to stabilize as the cells are expanded, and by passage 10 or so, there are no persistent epigenetic memories of what the cells once were.

Our iPSCs derived from cartilage and melanocytes are no better than other cell lines at making their cell type of origin, and they all make all three germ layers just fine.  The paper just out from Doug Melton’s lab shows that a simple manipulation in the culture conditions, just adding DMSO, makes both hESC and iPSC lines lose much of their bias toward a particular form of differentiation (Chetty et al, “A simple tool to improve pluripotent stem cell differentiation” Nature Methods, online publication 14 April 2013).  The message is that if we have robust differentiation protocols, all pluripotent cell lines will differentiate into whatever we wish them to.

4. iPSCs can also have various other distinguishing features from hESC ranging from subtle differences in the transcriptome and metabolomes to other areas. Overall, do you view hESC and human iPSCs as essentially “bioequivalent”? In other words, are they functionally interchangeable? In regards to the first 3 questions above as well, in the end is the bottom line how the cells behave above and beyond all else?

Jeanne: I think that the preponderance of evidence is that iPSCs and hESCs are bioequivalent.

I know that this is not yet the most prevalent opinion; attractive ideas are hard to shed.  Another recent example is the attractive idea that iPSCs will be rejected even if they are transplanted autologously (that is, to the identical strain of mouse, and by analogy, to the same person from which the iPSCs were derived).  One paper published in 2011 started this idea; two papers since then have refuted it very well. The good news is that if we’re patient, the real story eventually emerges.

5. What is your current preferred method for production of iPSCs for potential clinical use and why? The last time I talked to the FDA, they indicated to me (informally at least) that their experiences and regulations derived from oversight of hESC studies (Geron, ACTC) would provide a framework for their oversight of iPSC-based drug products assuming no genetic modifications invoked to make the iPSCs. Is that your impression as well?

Jeanne: There are two different ways to look at this:  what do WE think is the best, safest, method, and what does the FDA want us to do?  Unfortunately, the FDA will not tell us what to do, so we’ve decided, of course, to use non-integrating methods (Sendai or episomal vectors).

My personal view is that it’s also critical to do intensive molecular analysis of the cells: genome and epigenome- and correlate those data with the behavior of the cells.  For example, if we find that a particular duplication or mutation in the genome is consistently linked with bad behavior (like making tumors), then we can make sure that those cells are not used for therapy.

Stem cell genomics is one of my soapboxes:  we have all the tools we need for comprehensive genomic and epigenetic analysis of human pluripotent cells and their differentiated derivatives, and we should use them. My only caveat that the data MUST be interpreted biologically.  In other words, I would want researchers who are doing in depth analysis of stem cells to have great expertise in both genomics and in stem cell biology.

6. Can you tell us a little bit about your own lab’s clinically-oriented iPSC-based work?

Jeanne: We have active projects for both “disease in a dish” and transplantation therapy using iPSCs.  The work that is closest to a clinical application is our project to develop an autologous therapy for Parkinson’s disease.  A very long time ago I worked on fetal cell therapy for Parkinson’s disease; since the 1980’s, there have been multiple cases of successful transplants of human fetal dopamine neurons to PD patients.  Issues of fetal research aside, we think that our ability to quality control iPSC-derived dopaminergic neurons before transplant, which was difficult with fetal cells, will make this a much more consistently successful therapy.  Note that this work is fully supported by a patient group- Summit4StemCell.org. The stories of the 8 patients in the pilot study are on the web and are remarkable.

We have recently initiated a collaborative project on Multiple sclerosis that is supported by CIRM funding. Tom Lane of UC Irvine is working with my group to develop a stem cell therapy for this disease.

We’re also studying autism with a disease-in-a-dish approach, and have a small study on the possible role of stem cells in melanoma.  For the future, we have been developing an ethnically diverse collection of iPSCs that we hope will be useful in drug development, to identify drugs that are toxic to people with certain ancestries.

7. Finally, what is your perspective on the intellectual property (IP) area of iPSCs? I’ve been told that we should prepare for an iPSC patent war. Is that an exaggeration? More operationally and practically, does a  lab such as yours (or perhaps mine at some future date if we get to that  point with iPSCs) have to worry about who might own the IP rights to use iPSCs that your own lab might produce depending on how you make them?  How does a scientist navigate that legal and IP complexity?

Jeanne: Patents interest me.  Just last week, I visited the Supreme Court to watch the arguments about whether human genes should be patented.

Patents on fundamental things:  genes, human embryonic stem cells, iPS cells- allow the patent holder to have a monopoly, preventing anyone else from using whatever they’ve patented.

Patents are supposed to stimulate investment in development.  Why, as Justice Scalia said last week, would anyone have the incentive to study a gene and, for example, develop diagnostic tests, if they couldn’t prevent everyone else from working on that gene?

But patents also stifle competition and the advances that come from having many different groups studying the genes or cells.  One of the main reasons I returned to academia was so I could have freedom to study human ES cells without worrying about getting threatening letters from a patent holder, demanding that I either stop working on the cells or pay a steep licensing fee.

There will inevitably be problems commercializing iPSC-based therapies and assays, because at least three institutions own patents on aspects of iPSCs.  I’m paying attention to the patent “landscape”, but have decided to deal with those problems when they arise, and hope that the iPSC patent holders realize that the potential of these cells is too great to keep to themselves.  It would be better for all of us if the issue of stem cell patents never has to be decided in the Supreme Court.