From mature skin cells to dopaminergic nervous cells in animals

Interview  of Dr Vania Broccoli, Researcher at San Raffaele Hospital in Milan

First of all I should like to know who Dr Vania Broccoli is.  Where do you come from, what did you graduate in and how did you start working with stem cells at S Raffaele Hospital?

I come from Romagna, from Cesena to be exact.  I graduated in Biology at the University of Bologna, where I started to study nervous cell differentiation and the integration of the neuronal circuits  responsible for cerebral function.  After graduating I had the opportunity to go abroad to Munich (Germany) for three years, where I completed my PhD  in the field of genetic techniques applied to the central nervous system.  During my period in Germany I contributed to studies in mice that enabled the identification of genes that are indispensable for the development of the substantia nigra (Note: the area that is affected by Parkinson's disease) and learnt how to cultivate stem cells.    

Subsequently I returned to Italy and in 2001 S Raffaele Hospital in Milano employed me as researcher.  There I manage a biomedical research team within the stem cell research department, where other research teams work in other fields, including gene therapy.

The media have extensively covered the development of the technique to transform adult skin cells into dopaminergic nervous cells by your research team at San Raffaele Hospital, together with American researchers.  It is my understanding that the technique that "rejuvenates" adult skin cells, changing them back into embryonic stem cells, called induced pluripotent stem cells (iPS), was developed by a Japanese research team and that your discovery is the next step:  the technique to transform iPS cells into dopaminergic nervous cells - the cells that patients with Parkinson's disease lack.  It that right?

Yes, that's it.  The Japanese researchers lead by Prof. Yamanaka were the first to publish the rejuvenation technique.  They rejuvenated adult mature skin cells of a 60-year old man.  The team of Prof. Jaenisch in the US had been working on such a technique for some time and subsequently published work which confirmed the validity of the Japanese technique and improved it.  Now the technique is used in many laboratories throughout the world, including my own.  We all agree that it is simple and easy to use (all you have to do is inject 4 genes using a virus as carrier), and that it works not only on skin fibroblasts, but also on any kind of adult mature cell.  A dogma in biology has been challenged: contrary to common belief, it is possible to turn the biological clock of the cell back and rejuvenate it until it goes back to being an embryonic stem cell that is indistinguishable from those collected from embryos. 

How did you manage to transform iPS into dopaminergic nervous cells?

We took advantage of our experience in the field of embryonic stem cell differentiation and culture.  Experience is needed to apply the technique that we have developed.   Only experienced cultivators obtain rich harvests of cells ready for transplantation. 

There is a general issue I should like to raise.    There was the intention to ban embryonic stem cell research in this country on account of the ethical concerns related to their origin.  Now such research is essential for the exploitation of the cell rejuvenation techniques, which overcome the ethical obstacle.  Authorities should ponder their decisions regarding research bans, because one can never really know when apparently useless technology may suddenly enable a breakthrough.  Luckily, I had learnt how to cultivate stem cells in Germany. 

How long does it take to get a good harvest starting from the moment of collection of the skin cells to be rejuvenated?

A few months.

The transplantation of the dopaminergic cells was successful, wasn't it? What tests did you perform to prove its success and what were your results?

Yes, it was successful.  We used rats with a Parkinson-like movement disorder due to the administration of the toxin 6-hydroxydopamine, which destroys dopaminergic nervous cells.   After transplantation we observed clear improvement of motor symptoms,  on average after 4 weeks; the improvement persisted for the whole time of observation (8 months).  As rats live on average for 3 years, 8 months are like 20 years for a man, so it is fair to say that the results are satisfactory.  After 4 months some animals were sacrificed and their brains were examined.  We saw that part of the implanted cells had been integrated into the cerebral nervous circuits and that the improvement in motor function was proportional to the quantity of integrated cells.  This was a very important finding that shows that the improvement really was produced by the transplanted cells that had been integrated into the circuits. 

How many cells were transplanted and how many were integrated?

We collected thousands of dopaminergic nervous cells and 200,000 to 300,000 were transplanted.  It should be borne in mind that not all the transplanted cells were dopaminergic nervous cells; many were supportive glial cells, which contribute importantly to nervous cell function.  On average from 1,000 to 10,000 were integrated into the nervous circuits - thus, the survival rate is 1:30-300.

How did the co-operation with the American team start and what was your contribution?

I met Dr Wernig and Prof Jaenisch while I was working at Munich.  We remained friends and decided to continue our co-operation.  Our contribution was the development of the iPS differentiation technique and stem cell culture until a satisfactory harvest of dopaminergic nervous cell was available, whereas the Americans carried out the transplantations, monitored the animals and performed the autopsies.

These results were obtained in an animal model of Parkinson's disease.  What are the chances of getting a good harvest of dopaminergic nervous cells starting from iPS cells in humans?

According to me, the chances of success are high.  There is no reason to believe that human iPS behave differently.  We shall see whether this is the case soon, as we have started working with human cells.  I hope to have results to report soon. 

I know that there's another problem.  If I am not mistaken, the technique cannot be used in humans because one of the genes used for the rejuvenation process is an "oncogene" i.e. a gene associated with cancer.  How do you hope to solve the problem?   

The problem has already been solved, as we have learnt how to rejuvenate cells without inserting that gene.  

At what centre will the dopaminergic nervous cells be transplanted  into patients?

Nowhere at the moment. 

What, didn't you say that you think that the technique will work in humans and that the cancer issue has been solved?

Yes, I'm optimistic, but we have only discussed the positive findings, not the negative ones.  As all recent techniques (and this one was developed only 2 years ago!) it has some downsides, the first of which is, as you said, the risk of cancer.  Unfortunately some of the transplanted rats developed tumors.  We think that the reason was that we unknowingly transplanted undifferentiated iPS together with those that had differentiated into dopaminergic nervous cells and that the undifferentiated iPS started to grow out of control.  We have already developed a sorting technique for future transplantations, based on the identification of a protein expressed by undifferentiated cells, which disappears after differentiation.  Another problem is the risk of unpredictable effects.  For instance, a rat developed involuntary movements.  For this reason, before we transplant the cells into humans, we want to be able to control them and even kill them in an emergency. We already have an idea on how to achieve this and are working on it. 

Finally, there is the problem of cost.  Cultivating cells for months for only one patient involves very high expenses that would become enormous if the technology was offered to all patients with Parkinson's disease.  For this reason we are already thinking of a Bank with about one hundred already cultivated cell lines.  When a patient needing a transplant arrives, he or she would be given the cell line that is most compatible with his or her antigen profile. 

It seems that you have already thought everything through and that you have practically already solved all the problems.

Yes, that's why I am optimistic.

Dr Broccoli, thank you for your time. May I ask you for another appointment in a few months when you have the harvest of human iPS cells? 

Certainly, let's say in six months.  However, I think that I'll see you again before then. 

Really?  Where?

At the ICP Parkinson Institute in Milan.  As I already said, I truly believe in the potential of this technique.  Although it is too early to call in the transplantation surgeons, I think it is time for us to contact clinicians who see patients with Parkinson's disease, especially clinicians such as Prof Pezzoli who has experience with Banks of biological samples, namely the DNA Bank and the Nervous Tissue Bank.  I am coming to see him at the ICP Institute in a couple of weeks. 

I'll see you then.