Yamanaka’s baby turns 10 so here’s a top 10 list of IPS cell hot button bullet points

Shinya yamanaka

Wikipedia photo

Has it really been 10 years since induced pluripotent stem cells (aka IPS cells or IPSC) came onto the scene in the stem cell field?

Yes, it was a decade ago that now Nobel Laureate Shinya Yamanaka (山中伸弥) published that seminal Cell paper on reprogramming to make mouse IPS cells and then human IPS cells came the next year.

From the moment I read that first mouse IPS cell paper, I was very excited about the science and the ideas in it. The domain name of this blog The Niche is named after those remarkable cells, www.ipscells.com.

In honor of the 10-year anniversary, below I outline the top 10 IPS cell related questions and key points as of today looking to the future.

  1. IPSC and ESC as partners rather than competitors. Are IPS cells equivalent to hESC derived from leftover IVF embryos? Even if they are a bit different, does that matter? With both in the translational pipeline and available as the basis for research, we can achieve more as a field. Let’s see what develops. Will nuclear transfer ES cells (NT-ESC) ever fulfill the aspirational name of their production,” therapeutic cloning”? Or will they mainly be a cool, but somewhat esoteric tool for advancing knowledge and one used by only a few groups in the world? I hope there can be clinical impact from NT-ESC, but I’m very doubtful that it will become a reality any time soon.
  2. IPS cell trials. How will clinical translation of IPS cell-based products proceed in the next 10 years and sooner? How soon will the Takahashi study get back up to speed in its new form? Will other trials get going relatively soon (i.e. in the coming 3-5 years)?
  3. Diseases in a dish. Disease modeling using IPS cells continues to grow in importance. Will it continue to give the cell therapy side of IPS cells a challenge in terms of total positive translational impact from IPS cells? So far I would say disease modeling has had more impact, but that could change.
  4. Auto and allo. Autologous versus allogeneic IPS cell approaches are both generating buzz. As to the latter, what about those IPS cell banks in various places?
  5. Mutations matter but here’s the key context. Do IPS cell mutations matter? Of course they could, but most likely in the same way that ES cell mutations do. It’s more a question of genomic stability in general. What about mitochondrial mutations in IPS cells? The key thing here overall with genome issues is careful preparation and handling of cells and validating them rigorously. That doesn’t always happen.
  6. IPS cell sex. What about female IPS cells? Can we somehow “put an X” through the problems that sometimes appear associated with loss of X inactivation in female IPS cells? What about issues with imprinted genes? We don’t hear much about these things lately. As with the previous point, the bigger issue is validation of anything stem cell-wise that you’re studying, particularly if you have clinical intent down the road. Epigenomic validation more generally is very important for IPS cells.
  7. Patent big tent? Putting the IP in IPS cells or taking it out? Will there be any patent disputes of major significance moving forward or clinical research that is impeded by expensive licensing fees…or not so much?
  8. Directed direction. Is direct reprogramming going to heat up more so that it becomes a major alternative to IPS cells in certain cases? I hope so. The more cell types and methods we have, the better as long as they are supported by rigorous data.
  9. A vision for vision and beyond. Will the eyes continue to have it? Will IPS cell therapy development go beyond vision-related conditions soon? I’m sure it will, but eye conditions are dominant now as a focus for products made from IPS cells and ES cells. I can’t wait to see more trials for other conditions.
  10. Differentiation destination. In nearly all cases IPS cells will themselves not be used for therapies. Instead, differentiated cells made from IPS cells will be the actual therapeutic product. As with ES cells, a challenge with IPS cells is consistently making pure differentiated cells of the desired type. For instance, if you make 98% of say a neuronal cell type that you want and 2% of some undefined mesoderm or endoderm cells, that’s going to be a hurdle to overcome. The goal of cellular purity and specificity achievable with human pluripotent stem cell differentiation, but it can also be a real challenge.

Overall, I predict the IPS cell field will continue to mature and have even more impact in the next decade. A growing fraction of that impact will hopefully be coming from cell therapy-based clinical trials. There are likely going to be bumps in the road and even setbacks in the coming decade, but overall I’m very optimistic about IPS cells.

Dieter Egli Interview: NT-ES cells, IPSC, Mitochondrial Transfer, & More

Dieter EgliIt’s a particularly exciting time for the stem cell field.

One of the most notable developments in the last year or so is the production and preliminary study of a totally new type of human embryonic stem cells (ESC) made by nuclear transfer instead of using leftover in vitro fertilization (IVF) embryos.

This process of so-called therapeutic cloning has the power to produce patient specific ESCs called NT-ESCs that can in principle be used in the future for autologous transplants for a number of diseases including Diabetes. This gives the field three possible types of powerful pluripotent stem cells including NT-ESCs, IVF ESCs, and induced pluripotent stem cells (IPSC).

One of the leaders in the stem cell field and more specifically in the area of NT-ESC is Dr. Dieter Egli of the New York Stem Cell Foundation (NYSCF). His lab has published a number of exciting and important publications recently. I invited Dr. Egli to do an interview about the recent developments in NT-ESC, mitochondrial transfer (aka 3-parent technology), and the future of the stem cell field. I want to thank him for doing the interview.

PK: In your recent excellent Johannesson, et al. Cell Stem Cell paper you compared NT-ESC and IPSC. Were you surprised at just how similar they were?

DE: Yes we were. We thought that if there are differences, they should be visible in our assay, comparing genetically identical stem cell lines that only differed with regard to the method of their derivation. Previous work had drawn conflicting conclusions regarding the similarity of iPS cells and ESCs, but these analyses were done on genetically diverse stem cell lines. Both opinions had been voiced already, so this comes as a confirmation for some, for others as a surprise. It is indeed rather surprising that two very different paths to pluripotency, one resembling development, the other forcing a transition through cellular states that have no equivalent in nature, result in very similar cells. We must not forget though that such work cannot be exhaustive. The cells are similar with regard to the many things we looked at, but someone might want to look at their favorite process, to determine if there is a difference. We are happy to share the cell lines with anyone who wishes to perform additional analyses.

PK: Your findings are rather distinct from that of the Ma Nature paper from July from Mitalipov’s group. Can you comment at all on this difference and the possible reasons behind it?

DE: We had an advantage: we had two sets of nuclear transfer ES and iPS cell lines of adult and neonatal origin and had analyzed them using the same platform as Ma and colleagues.  As we finalized our manuscript, Ma and colleagues published their work, and made their primary data available to us as a result. So we were in possession of a larger set of data and cell lines. This difference was not entirely a matter of availability of cell lines, but also one of experimental design. There is a large amount of data on iPS cells available that could have been included in their analysis, to determine if their conclusions can be generalized. And some analyses are different between the two studies, for instance the whole genome bisulfite sequencing was not something we’ve performed, so we can’t speak to that.

PK: It seems like the big open question now is how the NT-ESC behave. For example, what are the differentiation properties of NT-ESC? Can you comment at all on this question of the functional properties of NT-ESC? Is it possible that NT-ESC could have some functional attributes that distinguish them from IPSCs?

DE: We don’t know the answer to this question. What I can tell you is that they both differentiate. There is of course great variability in differentiation efficiency of different human ES cell lines (e.g. Osafune et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol. 2008 Mar;26(3):313-5.) So it is not a simple task to draw solid conclusions. And there is nothing like the functional assays for human cells as there are in mice, where we can do tetraploid embryo complementation and ask if the stem cell-derived mice are normal. There will always be some degree of uncertainty regarding what we are working with in the culture dish. But I think we have just reduced this uncertainty, and now know that if iPS cells are made using methods such as modified RNA, they are likely very close to the real thing.

PK: What are your thoughts at this point as to the future of NT-ESC? Do you think they will have clinical relevance down the road as the basis for stem cell therapies or is that less likely now? In addition to possible translation to the clinic, is there more we can learn from NT-ESC and this kind of reprogramming?

DE: It is neither more nor less likely. There is no therapeutic application of reprogrammed stem cell lines yet. There are so many uncertainties that depend on biological, economical, and logistical factors, and on the patenting landscape, that it is hard to predict the outcome. With these many uncertainties, it would be risky to put all eggs into just one basket. I think the nuclear transfer field is going to have a strong presence and will remain a primary research focus of my lab.

PK: In addition to the studies mentioned above, I’m interested in the other work going on in your lab. Can you tell us about more about it? For example, do you have studies ongoing that are more focused on IPSC? Other areas?

DE: My lab is trying to understand beta cell dysfunction in different forms of diabetes. We are generating patient-specific iPS cells and differentiating them to beta cells to determine why these beta cells might fail more readily than those of controls. Genetic manipulations to correct or introduce specific mutations are now also part of our toolbox, but patient-specific stem cells are the primary resource. The Berrie Diabetes Center and The New York Stem Cell Foundation Research Institute have provided an ideal environment for conducting such research.

PK: A hot topic right now is so-called human mitochondrial transfer technology (known colloquially as “3-parent technology”) to try to prevent mitochondrial disorders. The UK seems on the verge of potentially approving the use of this technology before year’s end. What are your thoughts on this technology? Would you like to see if approved in the US as well? How would you respond to those (including myself, by way of disclosure) who are concerned about possible risks?

DE: The vote in Parliament in the UK is about removing the legal bars that prevent this technology from potentially being used. We might see a legal hurdle in the UK removed, one that we don’t have here in the US, but it is not equal to this being applied. The review of efficacy and safety is a scientific and medical matter that must continue independent of the political decisions. My biggest criticism of the research is that the number of studies in this field is still very small. We and others have shown that in principle the exchange can result in a complete exchange of mitochondrial genotypes. We also know that the resulting cells show normal mitochondrial activities. This is reassuring, but an important part is the numbers. How often can we do this and it still is exactly the same as the last time? These are the questions that matter regarding clinical translation, and we don’t have all answers yet.

I am aware that you have been rather critical of this approach and so I was hoping you would ask me this question. We are all recipients of genetic material from two parents, 4 grandparents, 8 great-grandparents, and so on. The sine qua non of human reproduction is new combinations of existing genetic material. The technique proposed will do exactly that, combine normal mitochondrial DNA that some of us already carry within us, with the nuclear genome of the egg cell. This alters the pattern of inheritance from a disease-causing to a normal mitochondrial genome, thereby preventing the disease. Opponents of this technique often deliberately – and despite better knowledge – draw analogy to genetic manipulation, such as done in bacteria, plants or animals. This is wrong, because mitochondrial replacement cannot alter the DNA itself, nor does it provide a path to it, nor will it allow predicting the physical features of a child any more than by natural reproduction, apart from the fact that we know that there won’t be mitochondrial disease. I have no doubt that if provided accurate information, most will welcome the use of this technology. I am on the side of advancing this research, and I appreciate that we will have the guidance of the FDA to translate this work cautiously and responsibly.

Regarding the 3-parent term: men do not pass their mitochondrial DNA to the next generation, unlike women. Nevertheless, I don’t think there is a dad who feels he is less of a parent than the mom for reasons being that his mitochondrial DNA is not present in his offspring. Therefore, I don’t think that contribution of the mitochondrial DNA is sufficient to claim parenthood, nor is the mitochondrial DNA necessary to be the (genetic) parent, and I believe the term mitochondrial replacement or mitochondrial donation is a more accurate terminology.  There is more to parenthood than contributing mitochondrial DNA and the above term is not a word that should be used to describe this process.

PK: What excites you most about the stem cell field now? Where do you see the field in say 10 years?

DE: I have been in this field for now 9 years. At that time, things like generating autologous pluripotent stem cells, or differentiating beta cells were just an inspirational concept. In just that time, they have become a reality. The use of autologous patient cells for curing diabetes still sounds like a dream, but it makes me think that we are likely going to see this happen.

What Does New Paper Mean for Future of Nuclear Transfer ES Cells?

NT-ESC versus IPSC

Advances in therapeutic cloning reported in the past year have been very exciting.

Somatic cell nuclear transfer (SCNT) can be used to produce very powerful human embryonic stem cells (ESC).

These new cells are called NT-ESCs for short. Neither embryos nor reprogramming factors are needed to produce human NT-ESCs. See herehere and here for discussions of the pioneering papers reporting creation of NT-ESC including the first paper by the lab of Shoukhrat Mitalipov of OHSU, which I called the stem cell event of the year for 2013.

Now that human NT-ESC are a reality, the big question is how good these cells are compared to existing alternatives. For example, can they compete with induced pluripotent stem cells (IPSC) in terms of clinical impact as a basis for regenerative medicine?

Because NT-ESC are extremely difficult to make and have other issues (more on that below), the general sense in the field is that NT-ESC have to be clearly better than IPSCs in some concrete way to be a major, meaningful clinically relevant advance. Otherwise, what’s the point of going to all that trouble to make them when IPSCs are relatively so easy to make?

Just a few months ago it seemed that NT-ESC might jump that high hurdle.

Mitalipov’s team published a Nature paper in July (Ma, et al) claiming that NT-ESC are demonstrably superior to IPSC. You read see my review of that paper here in which I was pretty excited.

However, now a new, very important paper from Dieter Egli’s lab just came out in Cell Stem Cell reporting a very different result than that of the Ma paper. The new paper (Johannesson, et al; see graphical abstract above) conclusively shows that NT-ESC and IPSC are extremely similar cell types. So Johannesson, et al say that NT-ESCs are not better than IPSCs. Drs. Mitalipov and Ma are authors on the new paper as well that seems to contradict their own July NT-ESC paper.

We are left with a dilemma.

What to do when a Nature paper and a Cell Stem Cell paper only a few months apart so strongly disagree?

What the heck is going on?

Preview piece by Alan Colman and Justine Burley accompanying the Johannesson paper in Cell Stem Cell discusses this puzzle. Colman and Burley call the wide difference in data between the two papers “troublesome” and “puzzling”. One possibility discussed is that the differences in data could be due to technical distinctions between the methods used in the two papers, but it’s hard to imagine how both results could be correct since they are so opposite in nature. The sense I’m getting from the grapevine is that the field is leaning towards thinking that IPSC and NT-ESC are largely equivalent, at least at this point.

The Johannesson NT-ESC paper is entitled “Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors”. This impressive paper has 4 key points outlined in its highlights section:

  • Isogenic human NT-ESCs and iPSCs were derived from the same somatic cell cultures
  • Human NT-ESCs and iPSCs show similar profiles of gene expression and DNA methylation
  • De novo coding mutations occur at the same rate in human NT-ESC and iPSC lines
  • Loss of imprinting occurs in both NT-ESC and iPSC lines at similar frequencies

In a number of assays reported in this paper, the IPSC and NT-ESC appeared essentially the same with remarkably similar rates, for example, of mutations and epi-mutations. Again, keep in mind that IPSC are a piece of cake to make compared to the challenges that go into making NT-ESC. It is reasonable to expect that making NT-ESC will get easier as more labs try the published protocols, but it is unlikely to ever be as simple as making IPSCs.

The other, non-trivial complication with NT-ESC is that for every individual NT-ESC line must be produced from a separate human egg obtained from a human donor, which invokes difficult practical and potential bioethical issues. For example, compensation for human egg donation is barred in numerous states in the US and in some countries.

NT-ESC technology has a third potential disadvantage of potentially paving the way for human reproductive (“Star Wars”) kind of cloning. This is definitely a dual-use issue for the stem cell field to consider carefully and openly discuss.

I’m glad to report that the proponents of NT-ESCs (e.g. see my interview with Mitalipov on this topic here) oppose human reproductive cloning, but that wouldn’t stop rogue labs from going ahead and doing it anyway, especially if the cloners can piggyback on key, published methodological steps from therapeutic cloning technology. You see, the exact same initial steps would be shared in the processes of human reproductive and therapeutic cloning (see diagram here). They diverge later.

So what’s the bottom line and where does this all leave us?

At this point, human IPSC and IVF ESC are far ahead in terms of clinical translation compared to NT-ESC, but it’s early days in NT-ESC research so I agree with Egli’s team that it is logical to continue research on NT-ESC. Let’s see how things look as we learn more about these cool, new cells, but at the same time let’s discuss all the implications (potentially positive and negative) and bioethical issues related to NT-ESC as well.

Cloning is cloned again: New Nature Paper is 3rd on Human SCNT

A new human therapeutic cloning paper is out today, the third in a matter of months. This one is from the lab group of Dr. Dieter Egli published in Nature demonstrating production of nuclear transfer embryonic stem cells (NT-ESCs) from an adult human somatic donor via somatic cell nuclear transfer (SCNT).

This human SCNT paper follows on the heels of a similar paper (Chung, et al.) from Bob Lanza’s group published in Cell Stem Cell and the pioneering Mitalipov human SCNT paper (Tachibani, et al.) in Cell in 2013.

Together these three papers have proven that human therapeutic cloning to make patient-specific ES cells is absolutely the real deal and that it presents a new therapeutic option based on stem cells in the years and decades to come.

This Egli group paper, Yamada, et al., is entitled “Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells”.

Yamada Extended Data SCNT

So what’s the scoop on this new Yamada human SCNT paper?

The main conclusions fit with those of the previous Tachibani and Chung papers. Oddly enough, one of the most important sets of data is tucked away as Extended Data Figure 8 (see above) that nicely summarizes the paper’s data.

There are some additional technical data that may prove useful for additional labs to make NT-ESCs by therapeutic cloning of human somatic cells such as surprisingly the inclusion of fetal bovine serum (FBS; see the figure above, the far right two columns showing that addition of FBS seems to really boost the process of making NT-ESC lines.

This team also made NT-ESC from a Type I Diabetic patient highlighting the future clinical potential of this technology.

What about the bigger picture?

As I mentioned in a previous post providing broader perspectives on translating human NT-ESCs to the clinic there are some key challenges and I list the top 5 hurdles. I called human therapeutic cloning to make NT-ESCs the stem cell story of the year for 2013.

It’s still a very big deal in 2014. The two new 2014 human SCNT papers just raise the intensity of this story to another level. It will be a fascinating story to continue to follow.

Top 5 challenges for SCNT cloned human embryonic stem cells

It was intriguing last week to read about another advance in somatic cell nuclear transfer (SCNT)-based therapeutic cloning of human embryonic stem cells (hESC). The first such work was published last year by Mitalipov’s group from OHSU.

Knoepfler Diagram Human Cloning

This second paper to produce so-called nuclear transfer hESC (NT-hESC) made the important advance to show that it could be done using adult and even old human somatic cells. This is a reproducible technology, which is very important.

However, key challenges and concerns remain for human therapeutic cloning and for potential clinical application of NT-hESC. Below is my list of the top 5 challenges.

  • NT-hESC must be indisputably better than human iPS cells and IVF hESCs to be relevant clinically: NT-hESC face very high hurdles. They must be demonstrably better in some key way than induced pluripotent stem (iPS) cells or traditional hESC made from IVF blastocysts or there’s no point in making them. If NT-hESC are only about the same as these other human pluripotent stem cells in terms of most key attributes then given the difficulty of making NT-hESC (even factoring in some anticipated improvements in technology) there would be little reason to make NT-hESC from a clinical perspective. Thus, their production would be limited to intellectual inquiries. While NT-hESC have the potential benefit of being used for autologous therapy (as opposed to IVF hESC being limited to allogeneic use), the other issues uniquely facing NT-hESC including some mentioned below make this trait of NT-hESC probably not enough alone to carry them forward.
  • The head start of other human pluripotent stem cells. Human iPS cells were first reported in 2007 and thus have a 7-year head start on NT-hESC.  The first clinical trial using cells derived from human iPS cells began enrolling patients in Japan in August 2013. Traditional hESC made from left over IVF blastocysts have been around much longer and their clinical trials started even earlier (ACT’s trials for MD). So in a sense NT-hESC could are starting far behind from a translational medicine perspective. On the other hand, one might say that the regulatory and scientific hurdles cleared by both hESC-based products and human iPS cell products might pave the way for NT-hESC and speed their translation to the clinic. Perhaps, but perhaps not. It’ll be fascinating to watch how this develops.
  • Human egg procurement challenges. The efficiency of making NT-hESC is very low. The legal and regulatory challenges of human oocyte procurement means NT-hESC production must either boost efficiency or find a new source material. For example in the latest paper only 2 lines were made from 77 oocytes. Some have said this is no big deal since the efficiency of making iPS cells is also inefficient. However, there’s a critical difference. When making iPS cells we start with proliferative somatic cells and can essentially use as many as we want (e.g. tens of millions), while in contrast when making NT-hESC each line must be derived using a separate human egg. Therefore, the efficiency of making NT-hESC must either be boosted at least say 5-10-fold or a substitute for human eggs must be found. In regard to the latter possibility, Mitalipov’s group has shown that at least in mice, two celled embryo cells can mediate successful SCNT.
  • The dual use dilemma for human cloning. One of the headaches for the advocates of NT-hESC is that potentially each advance in making NT-hESC (therapeutic cloning) could unintentionally also make it easier for some crazy folks to try to actually clone a person (reproductive cloning or “Star Wars” type cloning). Think that reproductive human cloning is impossible? Unfortunately, that challenge is not going to stop people from trying. Further, even failed attempts at human reproductive cloning (and it’s very likely the first attempts at human repro cloning would be horrible failures potentially producing deformed or dead humans) could unfairly, but rather quickly sink therapeutic cloning. I personally do not believe that there is any insurmountable technical obstacle to human reproductive cloning as it has worked for many mammals in the past and animal cloning is more common now than ever.
  • Cloning confusion and public opinion. Cloning is a confusing topic for the public. It is not always so easy for people to differentiate between therapeutic and reproductive cloning. Many folks may already think that “cloning” is bad as they conflate all types of cloning together. It is sort of like when people use the umbrella term “stem cells” to refer to all types of stem cells together. Unfortunately some of the people thinking in these overly simplistic ways are powerful political leaders. This remains a practical challenge for NT-hESC. Above is a picture from my book, Stem Cells: An Insider’s Guide explaining the differences between reproductive and therapeutic human cloning.