Retinal disease will get cell therapy first'

Interview with stem cell researcher Shinya Yamanaka.

SHINYA YAMANAKA, the Japanese physician and renowned adult stem cell researcher, says, Any new technology cannot escape from ethical issues. I think what is very important for us is to make our research transparent and visible to other scientists [and to] society. He spoke at length about his research on induced pluripotency stem (iPS) cells and cell-based therapy to Frontlines Science Correspondent R. Ramachandran. He visited India early this year to deliver a lecture on New Era of Medicine with iPS Cells as the featured speaker in the third edition of Cell Press-TNQ India distinguished lectureship series in Chennai, Bangalore and New Delhi. Excerpts from the interview:

Is the molecular mechanism of reprogramming [of adult somatic cells into iPS cells] fully understood?

Since thousands of factors contribute to reprogramming, the molecular mechanism of reprogramming is hardly understood until now.

Does the fact that you are able to generate iPS cells from somatic cells indicate that somatic cells preserve some kind of memory of their embryonic stem (ES) cell origins that they are able to be reprogrammed back?

Oh! I see what you mean. Mmmm That may be the case, but in my model it is kind of opposite. Somatic cells have to completely lose their memory as being somatic cells. I think of ES cells as a white board and cell differentiation is, in my view [Yamanaka emphasises], a process to write many different pieces of information on that white board. What we are doing by iPS cell generation is to erase that information, make it white again. Fertilisation of sperm and egg is a kind of erasure that occurs normally. It is very rapid. Within 24 hours all the memories have gone; they become completely white. I believe that is because after fertilization many proteins that function as erasers play different roles to make it completely white. Each factor probably erases [a] different part. So it is a very nice team work. But we only identified four factors, four erasers. Usually this is not enough. Four erasers can only erase some portion. But since we are introducing many of them by retroviruses, since we are over-expressing those four erasers, they will start erasing where they normally do not erase. It is like erasing the board white by closing your eyes. It takes time. But after about 20-24 days, the board can become almost completely white. So it's kind of a stochastic process.

What are the chances that iPS cells retain some of the memory because one is using only a few transcription factors and there is no total erasure that could affect any kind of directed cell transplantation therapy one might be considering?

Oh! I see. You know some scientists are arguing that iPS cells may retain some memories of original somatic cells that may affect therapies like this. It is still controversial.

Apart from carcinogenicity there is also the issue of mutagenesis due to the genomic integration of genes from the retroviral vectors used for transfection. Do you see these being key hurdles in therapeutic applications of iPS cells?

Yes. Safety issues like carcinogenicity is perhaps the most important and hardest hurdle that we have to overcome, but I think we are getting closer [to getting there]. You know we have overcome the issue of c-Myc and the issue of retroviruses' DNA integration [into the host genome]. We can now generate iPS cells without any DNA integration by using plasmid DNA. Once established they are completely free from any transgenes. Actually, you could use even c-Myc to make iPS cells. Because once they become iPS cells they do not have any c-Myc transgenes. But to be on the safer side we do not use c-Myc.

So the possibility of mutations arising from genomic integration, which was the other apprehension, will also not be there. Is that right?

We can, of course, now avoid genomic integration. But we may still have point mutations. But I believe even ES cells should have some kind of point mutations. I think after culture in a dish no type of cells can be free from such mutations. I don't think any [cultured] cells can be 100 per cent free [of mutations]. So the question is to what extent we can say it is okay. That's what is important. It's just like any car or any airplane. If you perform very intense examination using a microscope I am pretty sure that you will find some kind of small defects. But if we cannot find any by eye examination and that's what we have been doing with cars those cars are just fine. So if we identify very huge variations in the iPS cells, we should not use those. So the question is how much we can accept and how much we cannot. That's what nobody still knows.

But you perhaps know the extent of point mutations in ES cells. There must be sufficient data on ES cells to give you that information.

Well, actually we still do not have enough data because full genome sequence became available only very recently. It is less than 10 years since the first human genome was sequenced. But now we can sequence one full genome in 10 days. So what we are now trying to do is to sequence each iPS cell clone completely and to pick up iPS cells with the least number of mutations and then test those cells in vivo to confirm that those cells do not form tumours.

But I would imagine that you will still need to work with ES cells for comparison to know how much of mutation is acceptable.

You are absolutely right. ES cells are the control for our studies. We have to continue using human ES cells.

So the regulations on the use of ES cells and the question of how many ES cell lines are available for research, etc. become relevant.

We have been using 10 human ES cell lines. For the moment it is sufficient for us. We do need human ES cells for control.

Returning to the issue of genomic integration, how does reprogramming work without such integration? How do the ES cell characteristics get transferred to the transfected somatic cells?

We have been using the so-called episomal plasmid systems. They can replicate in transfected cells for about two weeks. But once they get converted into iPS cells, since iPS cells proliferate very fast, the replication of those episomal plasmids cannot catch up. They spontaneously disappear from the cells. So it is very convenient.

Since the transfected genes are not fully integrated into the DNA, is it clear that these cells will perform all the functions properly once you transplant the differentiated cells into the body?

Oh! It's okay because once they become iPS cells the endogenous factors are activated. So we don't need transgene expressed exogenous factors anymore.

But is there any optimum level of exogenous factors for the required over-expression that triggers the formation of iPS cells and the activation of endogenous factors?

Yes. If in the process of conversion of somatic cells we do not have sufficient amount of exogenous factor, they cannot become iPS cells. If they have too much of Oct4 they cannot become iPS cells. So only a small proportion of fibroblasts or blood cells in which the four transgenic factors are expressed at the right level, only those cells can become iPS cells. So it's a kind of natural selection.

Is it why the conversion efficiency is so low?

It is certainly one of the reasons. Yes. I believe so.

How can one improve this efficiency?

Well. It is low. But from a practical point of view it is sufficient. From a scientific point of view, the efficiency is very low [0.01 percent]. From each experiment using one small cell culture Petri dish we can make 100 independent iPS cell clones from about one million fibroblasts. But from a practical point of view, I think the efficiency is high enough as 100 iPS cell clones are more than what we need because we need only a few and these will proliferate.

But then it raises the question of how good and how pure those 100 iPS cells are.

That's what we have been working on. And those 100 iPS cell clones are not identical although they are from the same donors and from the same experiment; they are still different. Some have complete reprogramming and some don't. So we really have to choose the right ones.

So, as you mentioned in part I of the interview, besides the teratoma formation you have to look for other characteristics of these iPS cells.

Yes. Such as markers, gene expression analysis, DNA methylation analysis with DNA micro-arrays, etc.

Coming to the question of cell-transplantation therapy, how do you home these cells to whichever target they are to be directed when you inject them into the body?

Well, that depends on each type of application. First, of course, we have to induce the iPS cells to differentiate in vitro and these have to be purified to the required type. Then these purified cells have to be introduced in vivo at the appropriate target.

For example, in retinal diseases such as age-related macular degeneration [AMD, a medical condition which results in loss of vision in the centre of the visual field (the macula) because of damage to the retina and usually affects older adults, but sometimes younger people too] what we are trying to do is to first make retinal cells from iPS cells, the retinal pigment epithelium [RPE] cells.

Then we make a sheet of RPE cells. Then we actually transplant these cells underneath the patient's retina. We just do that by a physical procedure. And in the case of Parkinson's disease we will inject dopaminergic neurons in a very specific site of the brain where these neurons normally exist. So we have to do a kind of surgical procedure. The transplantation in patients is, therefore, as cells by themselves or as a sheet.

You mentioned experiments where adult stem cells of one type can be transformed in vivo into another type, like Drosophila eye cells turning into leg cells. Does this have to do with the niche environment in which stem cells of a given type normally reside and differentiate according to that specific environment but one is not able to provide that kind of environment in vitro? Does that in any way affect the procedure?

Yes. As a matter of fact it is still very difficult to make functional beta cells of the pancreas or hepatocytes [main cells of the liver tissue]. But we can make pancreatic progenitor cells from ES and iPS cells.

These progenitor cells are still like stem cells; they cannot produce insulin. But several researchers have shown that, if you transplant those pancreatic progenitor cells into the pancreas, the right niche, they can differentiate into beta cells in vivo and then start producing insulin. So it is a kind of combination of in vitro directed differentiation and in vivo differentiation.

Shouldn't that kind of approach be more suitable for all kinds of differentiated iPS cells? Or is this unique only to some kind of differentiated cells?

I think it is unique only to some applications because once we transplant cells in vivo, we cannot control them. But in vitro we can observe, we can normally check easily and we may also be able to tune those cells if something really bad happens. But once in vivo, we cannot do anything. So [that approach] may be very dangerous.

But do we understand the entire signalling process the whole chain of biochemical signals required for the process of differentiation so that you can intervene appropriately?

No. No. That is what many researchers have been working on. Usually to induce in vitro directed differentiation we try to follow what nature does during the differentiation process; like, for example, pancreatic development is a very, very complicated process. It takes a very long time. At each stage different signals cytokines, growth factors play a role.

So many researchers have been trying to identify those multiple stages in normal development and then try to recapitulate [replicate] those in vitro by adding different cytokines sequentially or something like that. But it's still very difficult. Of course, it is very difficult to beat nature.

When and how do you actually induce the in vitro differentiation of these iPS cells into specific cell types?

iPS cells grow indefinitely but stop growing once they start differentiating into functional cells. In general, when researchers culture cells, the cells are periodically stored in a frozen state to stabilise their characteristics after the passage of certain time. But we are yet to find out the best timing of differentiating iPS cells into functional somatic cells.

Once you inject the differentiated cells into the human body, how long-lasting is their capacity to regenerate? That is, if we do cell transplantation therapy in a person, will it be long-lasting or short-lived?

Well, we know from experiments on animals that they can survive at least for a year or two. But we don't know more than that because we need much longer observation. So it will take some time. We may have to repeat cell transplantation after some years.

Is there a minimum amount of these differentiated cells necessary for effective cell therapy? Is there also a risk arising from an overdose of these new artificial cells?

We need to do further study to find the best amount of cells for transplantation for each disease.

ETHICAL ISSUES

Some experiments have suggested the possibility that we may be able to generate embryos from iPS cells.

Well, in theory at least we could generate sperms and oocytes [egg cells] from iPS cells. So if we perform [ in vitro] fertilization at least in theory we may be able to generate new life from skin, for example. Of course, technically it is still very, very difficult. I don't think it's possible. But at least in theory it may be possible in the future. So it's kind of ironic. To make iPS cells we don't need human embryos. So it is free from ethical issues. But in the future those iPS cells can make new life. So we may be facing new ethical issues.

So what kind of ethical guidelines and regulations exist with regard to the use of iPS cells in various countries, including Japan?

In Japan we are now allowed to perform experiments in which we try to generate germ cells from iPS cells. However, we are not allowed to perform in vitro fertilization of those sperm and oocytes from iPS cells. That limitation we have.

But isn't the earlier process used to create Dolly the sheep, namely cloning through somatic-cell nuclear transfer (SCNT), now possible with iPS cells?

It's possible. Because nuclear transfer is very difficult [and] technically very challenging. The success rate is very low. However, they have shown that if you use nucleus from ES cells the success rate is much higher. So that means that if you use nucleus from iPS cells, the success rate will be equally higher. So you are right. By combining the two technologies, nuclear transfer could become easier.

So the same old issues which arose around Dolly would again come up

Yes.

So would you agree that while one was getting rid of one set of ethical issues we could be faced now with a whole new set of ethical issues?

Yes. Yes. Any new technology cannot escape from ethical issues. I think what is very important for us is to make our research transparent and visible to other scientists [and to] society. We should not hide anything.

Coming to the other aspect of your iPS cell research, namely, disease modelling which you mentioned in your talk. How do you go about it and what is the current status of the research?

Well, that is a more promising approach in the context of this technology. You can easily imagine that it is very easy to make models of genetic diseases caused by one mutation because iPS cells can retain the same mutation and we can easily recapitulate [replicate]. And that is very good. But at the same time we initially thought that it will be difficult to recapitulate [replicate] diseases such as amyotrophic lateral sclerosis [ALS] or Alzheimer's disease because these are late onset diseases. Until 40 or 50 years [of age] those patients are just fine. It is 40 years!

So we thought it should be very difficult to recapitulate [replicate], to make disease models of those diseases with iPS cells. But, as I briefly introduced [in the talk], many researchers have now shown that even [in] diseases such as ALS, Alzheimer's and even schizophrenia, neural cells derived from iPS cells are abnormal already. So I think in vivo, in patients, there are multiple compensating mechanisms.

The neurons are protected by other types of cells in their niche [environment in the body]. That is why it takes so long before patients actually realise the symptoms. But in Petri dish those neurons are very lonely. They are not protected by anything. So I think that may be the reason why we see some differences from the beginning [itself]. But it is different from the real state or patients. So it is kind of a very artificial model. But still it's a human cell. It's the patient's own cell. It's not perfect. That's for sure. But it is certainly an unprecedented way to analyse human diseases.

You in fact mentioned that motor neurons do not divide very well for modelling purposes and for that iPS cells are very useful as they proliferate fast.

Even motor neurons derived from iPS cells cannot divide. So once we make motor neurons [from iPS cells] and once we purify, they can survive only for a week or so. However, before that we can expand iPS cells to a huge amount. Then we can make a large number of motor neurons, which we cannot obtain from patients. Similarly, for many other types of cells such as cardiomyocytes and blood cells.

You also mentioned that there was no cure for ALS because there was no model for the disease. Has there now been any progress with the use of iPS cells on that front?

Yes. We do have those motor neurons from patients' iPS cells and we do see differences. By performing DNA micro-array analysis and DNA methylation analysis we now have a clue why those patients' motor neurons show those abnormal phenotypes. So we are able to get much more information now about the causes of the disease. At the same time we are now performing drug screening and we have already identified one drug which seems to be effective at least in vitro. By adding that chemical we can revert abnormal phenotype back into normal phenotype.

Is this abnormality in morphology only or in any other characteristic as well?

Morphology and also gene expression. But you know we are not so optimistic to say that this chemical should be effective in patients. It is important [to remember] that [only] if we are extremely lucky this chemical may turn out to be effective in patients. I think we just have to continue drug screening and if we identify something like 100 drug candidates using iPS cell-based assay we hope that one of those candidates may turn out to be effective in patients. So that's our strategy.

So what are the prominent diseases where you see the potential of identifying drugs?

Many. Such as ALS, Alzheimer's disease and other neurogenic diseases.

Each one of those diseases requires hundreds of chemicals to be screened. Are there any drug candidates already in the stage that they can go into the market soon?

Oh! No. Not yet. We are just beginning. Hopefully in the near future we should be there.

But pure cell-based therapy is probably a long way off.

It's a long way off and target diseases are very limited. Target diseases should be caused by loss of just one type of cells, as in retinal diseases, spinal cord injuries, Parkinson's and Type I diabetes. So these are very limited. However, drug screening can be tried on many multi-systemic diseases as well.

I recently read that in Japan iPS cell-based treatment of retinal disease is expected to go in for clinical trials soon.

Yes. Either this year or next year. We have quite [good] progress there. What is the beauty about retinal disease in terms of first application of iPS cells is that we don't need a huge number of cells. We only need something like 10,000 retinal cells and those are pigmented cells. So once they become fully differentiated we can easily detect those cells through the microscope because they should be pigmented. So, actually we will examine every cell before transplantation. And if we find some bad cells, which do not have pigmentation, we will kill those cells by laser. And after transplantation we can easily observe what is going on in patients by normal optometric techniques because it is there. Also, if we find something bad going on inside the eye we can kill those cells by laser. That's why I think retinal diseases such as macular degeneration will find the first application of cell therapy using iPS cells.

You mentioned that in the case of spinal cord injuries, cell transplantation should be carried out within a month and because of that patient-specific cell therapy was not feasible in this particular case. Is there a similar time limit for other target diseases as well? For example, in the planned trials for retinal disease, will it involve patient-specific iPS cells?

In the case of macular degeneration, patient-specific cells can be prepared, generated from the patients' iPS cells, because currently available drugs can only delay the progression of the disease.

You mentioned that you have had some success with mouse models for some of these target diseases.

We have made some progress.

You also mentioned that you will be soon launching trials of iPS cell therapy for spinal cord injuries. How long will that take?

I expect that the first clinical trial on a spinal cord injury patient may start in several years if our study goes smoothly without any major problems.

Since in this case you cannot have patient-specific iPS cells, do you plan to use cells from human leukocyte antigen (HLA)-matched donors? Even so, is there any risk of immune rejection? (To avoid immune rejection compatibility between the somatic cell donor and the recipient is important and this is determined by comparing their tissue types, also known as HLA types.)

We expect that if we can use iPS cells that match a patient's HLA type, we can reduce the risk of immune rejection as much as possible. It is also more desirable if the HLA-typed donor is also homozygous. [If the copies of each gene, one from the father and one from the mother (referred to as the alleles of a gene) are identical (in terms of mutations or copying errors), the individual is homozygous.]

What are the chances of something going wrong in vivo in such cell transplantation therapy despite all the purification that one may do in isolating fully differentiated cells, say from residual contamination or some other factor? Can one ensure 100 per cent purification?

It's always possible. You are right. We have to double check at each step to ensure that it is correctly done. At the initial stage we have to check complete reprogramming [of iPS] cells and in the second stage purify the cells we want. That is why we feel very safe with retinal disease where we can be 100 percent sure. We can check each individual cell and we can easily kill [unwanted ones] before transplantation. In case of spinal cord injuries and diseases such as Parkinson's we really have to be very careful. We have to transplant more, like more than one million cells. Well, we can check by flow cytometry to ensure that they have zero per cent of undifferentiated cells. We have to perform animal safety tests before any human application. But that is what the American company GERN [Geron Corp.] did [before it proposed Phase 1 clinical trial on humans which was approved by the Federal Drug Administration in 2009]. Unfortunately, it stopped doing that [in 2011 for financial reasons]. The company proposed to use ES cell-derived oligodendrocytes [a kind of brain cell] in spinal cord injury patients. It had to [first] test in rats and mice.

You mentioned two other companies one ReproCell in Japan and the other Cellular Dynamics in the United States in your talk. Have they also been licensed to do such trials?

No. They only sell cardiomyocytes from iPS cells for pharmaceutical purposes. Not for transplantation. Well, it is too early [to do myocyte transplantation.]

Have any new drugs been identified through drug screening studies using cardiomyocytes from iPS cells that were not known before?

Yes. As I mentioned, they have been using the so-called hERG [safety] test to predict drug action on cardiomyocytes. Some researchers have confirmed that some drug candidates were dropped because of hERG test but they showed that the test was false positive. So the drugs need not have been dropped. As I mentioned, it is so artificial that you can easily have false positives and false negatives. Both are not good. So it is too early. Drug discovery is a long, long process.

You mentioned that originally you obtained somatic cells by the technique of skin punch biopsy, but more recently you have developed a technique that avoids this and uses only peripheral or circulating blood. In terms of efficiency how good is that?

Compared with skin fibroblasts it is less efficient. But again, from a practical point of view it is good enough. So I think we will use peripheral blood or [umbilical] cord blood. You know many countries have cord blood banks. So we may be able to collaborate with those existing cord blood banks.

You started out doing basic research with a clear goal of being able to treat intractable and incurable diseases and improve the lives of people. But normally one does basic research without such an application-oriented goal in mind. You seemed to start out doing basic research with a different kind of philosophy.

Not really. Again it is a very long process from basic research to clinical translation 10 years, 30 years, 40 years. I really wanted to be just one scientist in that long, long process. I wanted to make some progress, some contribution. I didn't think that I could do everything.

But you did say that you set yourself the goal of producing ES-like cells from somatic cells. You seemed to be clear in your mind that this goal was achievable. I was quite struck by that remark. And you have really achieved that.

Well, what I learned during my early training in research is to be that way vision and hard work. You must have clear vision. But even that process of making ES-like cells is very, very difficult. It is still only a small part of a long, long process. But we are getting closer to the clinical stage.

I really want to make it as short as possible. Hopefully, we can reach the goal before I die.

Professor Yamanaka, thank you very much for your time.