Making functional bone marrow from ES cells – Dr. Nicholas Zavazava interview

by Alexey Bersenev on February 28, 2008 · 1 comment

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Nicholas Zavazava, MD, PhD – Professor of Internal Medicine, Division of Allergy/Immunology. Director, Transplantation Research, Department of Internal Medicine, University of Iowa, USA.
This interview was taken according his group recent study, prepublished in Blood journal.

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Dear Dr. Zavazava,
I’d like to thank you for the great solid study done by your team, prepublished in Blood journal, and for this interview.

1. The first question is: why is Embryonic Stem Cell (ESC) an alternative source for hematopoietic progenitors? As we know, other traditional sources (such as bone marrow, cord blood and mobilized peripheral blood) can provide us with good results in bone marrow transplant (BMT) clinics. The problem of immune incompatibility could be solved by using autologous or partially matched transplants from relatives and improvement of inter-hospital HLA database network. The problem of low HSC number in donor marrow could be solved by combining a few samples (for example, this approach is used for cord blood) and enriching for HSCs or expanding HSCs ex-vivo. So, in short, what advantages over these other methods do ES cells provide that make them a source of HSCs?

NZ: ES-derived cells are less immunogenic than any other cells you mentioned. They grow much faster and can generate all hematopoietic cell lineages. Thus, treatment of patients could potentially be much safer and the hunt for an HLA-matched donor might be avoided.

2. The next question follows from the previous one: If we are talking about making this technology more practical than bone marrow transplantation, where should this embryonic stem cell come from? Should it be a patient-specific or already existing cell line? Is it clinically proven that embryonic stem cell lines do not cause immuno-rejection? Once adult stem cells are derived from embryonic stem cells, would they express HLA that will cause immuno-rejection anyway?

NZ: Of course we need more detailed experiments to confirm our presumptions. So far, our unpublished data appear to point in the direction that even though the ES-derived cells later acquire the ability to express MHC antigens, that gradual expression of these antigens is better tolerated by the immune system.

3. I was wondering about how only ES cells transduced with HOXB4 gene survived and differentiated to HSCs in the medium with hematopoietic cytokines compared with non-transduced ES cells, of which 90% died by day 20 of culture. Is HOXB4 essential for hematopoietic differentiation in mice? Do you know other genes which could promote hematopoietic differention from ES cells specifically, and do the same genes control hematopoiesis in humans?

NZ: HOXB4, in our hands, appears to confer the ability for self-renewal and not necessarily the differentiation process. The processes are however intertwined such that we cannot make conclusive statements about that. However, we can differentiate non-HOXB4 transduced cells too, but we cannot maintain them in culture long-term as they quickly die away.

4. How can you explain that in the human system, which used the xenogeneic transplantation model, engraftment of HSCs derived from ES cells was not dependent on ectopic HOXB4 expression? Interestingly, some reports have found that HOXB4 promotes hematopoietic differentiation and expansion of human HSCs in vitro.

NZ:Well, those data are still conflicting in humans. However, it is possible though that the same gene may work differently depending on the species. More work needs to be done on human ES cells though.

5. Can you tell us anything about the molecular basis of how HOXB4 enhances ES cell differentiation to HSCs and how it keeps from going out of control?

NZ: HOXB4 is a homeobox transcription factor that signals proteins further downstream. The exact sequence of proteins being activated is not yet known. That detailed work on signaling is very expensive and will require dedication to these mechanistic studies. For now we cannot yet put together the mechanism. We do know that the gene is effective in hematopoietic cells and in ES cells.

6. Do you think it’s worthwhile to test the functionality of immune cells derived from ES cells in a human system? It could be a very interesting next step.

NZ: Absolutely, our work is going in that direction.

7. Question about safety issues. I think we always have to be alarmed when any gene introduced to and overexpressed in cells is transplanted into humans. Do you think that overexpression of “hematopoietic” genes potentially carries a risk of myelo- or lympho- proliferative disease development?

NZ: Yes I do. However, experiments in mice and primates have shown that it is safe. The caveat is that these animals have much shorter lives than humans, so we simply do not know. What our data and those of others do at this point is teach us that we can indeed get large quantities of the HPCs under the influence of HOXB4 and what we need to do is use this system to better understand its influence on cell self-renewal and perhaps develop safer ways to get to the same result. We have started to use recombinant HOXB4, which does not need transduction of cells or introduction of another gene in a cell. If that proves to work, then we would have a protein to work with. It appears though that the transduced cells obey the control mechanisms in vivo preventing uncontrolled cell growth.

Indeed there are other growth genes out there that could be tested. But we will still have to answer the question of how do we avoid tumors after gene transduction.

On the other hand, we know that ES cells themselves or their derivates have a risk of tumorogenesis. In this case, in terms of clinical settings, should we make sure that there is no contamination of transplant by ES cells? Could this be one of the disadvantages of using ES cell-derived hematopoietic progenitors, or do you think it’s safe therapy?

NZ: Purification of cells is done anyway when using bone marrow stem cells. So that step is not problematic.

8. In your study, the mouse ES cells demonstrate good homing when transduced with HOXB4, but a quite similar (“Canadian”) study published in the Journal of Experimental Medicine in 2004 found that human cell homing was independent of HOXB4 expression. Given this inconsistency, how can the results from your study be translated to human cells?

NZ: The homing of these cells has not been well studied. That study you cite is only in my mind preliminary. We need more rigorous studies to answer that definitively.

9. According to the recent interest in iPS in stem cell research, what will be the role of iPS in this technology? Is it really good, and could it possibly be a clinical-grade approach?

NZ: Yes it could be. It would also be much safer. What is missing with the iPS technology are long-term in vivo studies of these cells. If we can show that they indeed can do the same and live long enough, then we will have an excellent alternative.

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Interviewers: Alexey Bersenev, Joanna Balcerek, Jae-Won Shin

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