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Eur J Cardiothorac Surg 2004;26:S54-S56
© 2004 Elsevier Science NL

Review

Implication of therapeutic cloning for organ transplantation

Institut für Neurophysiologie, University of Cologne, Robert-Koch-Str. 39, 50931 Köln, Germany

^* Corresponding author. Tel.: +49 221 478 6960; fax: +49 221 478 6965. (Email: j.hescheler{at}uni-koeln.de).

	Abstract

Top Abstract 1. Introduction Appendix

 
Replacement of damaged myocardium with electrically functional, contracting syncytium with a balanced blood supply remains a key goal for the treatment of hearts damaged by coronary heart disease or other disorders. Stem cell therapy offers a potential solution. This paper describes the value of in vitro stem cell research to unravel the roles of key regulatory molecules in embryogenesis of myocardium and blood vessels. Studies have shown that functioning myocytes can be derived from stem cells in vitro and engrafted into infarcted areas of heart where they develop into functional adult like cardiomyocytes with action potentials and capacity for beta adrenergic and muscarinic regulation. Further studies have identified specific roles for platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the sequential differentiation of blood vessels and capillaries.

Key Words: Angiogenesis • Cardiomyocyte • Embryonic stem cells • Vascular endothelial growth factor • Platelet endothelial cell adhesion molecule • Fibroblast growth factor

	1. Introduction

Top Abstract 1. Introduction Appendix

 
Because of their ability to reproduce the embryological differentiation of nearly all different cellular phenotypes, embryonic stem (ES) cells represent an ideal tool to study processes of embryogenesis under in vitro conditions, in particular the signalling cascades and genes involved in the functional development (functional genomics) as well as providing a new source for cellular replacement therapy. We have cultivated ES cells in three-dimensional cell aggregates, where they differentiate into derivatives of all three germ layers.

1. Embryonic vascularization comprises different processes such as proliferation, migration, differentiation and tube-formation of endothelial cells. We have established stably transfected mouse ES cell lines, where the endothelial-specific platelet endothelial cell adhesion molecule (PECAM) promoter drives the expression of the live reporter enhanced green fluorescent protein (EGFP). This approach enables investigation of morphogenetic changes and related signalling cascades in endothelial cells during early embryonic development. Morphogenetic changes of endothelial cells in the presence of key regulatory molecules fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are monitored employing time-lapse microscopy. At first, clusters of angioblasts are predominant which later develop into elongated network-like structures. VEGF induces proliferation and pronounced sprouting of angioblasts whereas FGF leads to stabilization of preformed endothelial structures. We conclude that the ES cell system in combination with endothelial-specific EGFP expression is a valid tool to investigate early events of vascular differentiation in vitro. VEGF plays an important role for early vessel formation whereas FGF is crucial for endothelial survival. Because of their ability to promote capillarization and angiogenesis ES cells may be useful for recapillarization after cardiac infarction.

2. This potential is paralleled by the ability to generate cardiomyocytes in vitro for tissue repair. Cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild type mice. Immunological cross-reactions were avoided by using the same inbred mouse strain. To allow identification of the transplanted cells transgenic ES cells were used carrying an IRES vector with two cloning sites for EGFP and an antibiotics resistance for selective selection both under the -MHC promoter. EGFP positive transplanted cardiomyocytes could be easily detected in the native heart at different intervals after operation. The cells were found to engraft and differentiate into adult-like cardiomyocytes as confirmed by cross-striation after immunostaining with -actinin. These data were corroborated by patch clamp experiments on isolated EGFP positive cardiomyocytes at different time points after operation. The transplanted cells displayed ventricular action potentials and ß-adrenergic as well as muscarinic regulation. When survival was investigated in a large colony of transplanted—(n=99) and control mice (n=36) where NaCl instead of transgenic cardiomyocytes were injected, the control group had an almost double mortality rate. Our data show engraftment and differentiation of embryonic cardiomyocytes after transplantation into cryoinfarcted areas of heart.

3. Before a clinical use of human ES cells for therapeutic trials in humans, two major prerequisites must be fulfilled: (i) they must be safe, i.e. the development of tumours because of the high proliferative potential must be omitted and (ii) the rejection of the transplanted cells must be prevented. For the criterion (i) the technology of ‘lineage selection’ has been developed, strategies in order to only allow the needed cell to differentiate from ES cells but all other cells are prevented to survive. This can be obtained by modifying the culture conditions and/or adding a combination of various growth factors and signalling molecules which preferentially supports the growth of a specific cell type, but prevents the development of other types. However, up to now, the transgenic drug selection approach has proven to be the most specific and effective for reliable purification of ES cell derived cells. For example, IRES-vectors encoding EGFP and the puromycin resistance gene under specific promoters are used. Application of puromycin (optimal time point will be seen from the EGFP expression) will eliminate all other cells except those containing the resistance gene. In our experiments using cardiac-specific promoters we found an extremely efficient purification of over 99%. For the criterion (ii) several suggestions have been made: the most simple one is of course the development of a stem cell bank containing 1000 or more ES cell lines with different immunological determinants (MHC surface complexes, HLA system). Alternatively it is suggested to use the so called ‘therapeutic cloning’, i.e. the exchange of the haploid nucleus of a donor oocyte by that of the patient. It has been demonstrated that the oocyte with the new nucleus develops to a normal blastocyst from where a new patient identical ES cell line can be prepared from the inner cell mass. In order to prevent the high number of donor oocytes it may be also possible to differentiate oocytes by the method introduced by Schöler.

	Appendix

Top Abstract 1. Introduction Appendix

 
Conference discussion

Dr D. Birnbaum (Regensburg, Germany): Probably my question is naive. We saw one picture where the area where the stem cells were injected took part in the contracting process according to the echocardiogram, which of course is a subjective interpretation. How can you be sure that this area is vascularized?

Dr Hescheler : That is, of course, a good point. Up to now we have injected only cardiac precursor cells. It should be mentioned that there is an interesting feature of these early cardiac precursor cells, i.e. they are completely resistant to hypoxic conditions, which might be due to the fact that the early embryos are in an environment with low oxygen. So embryonic cells may survive through an anaerobic metabolism. Furthermore they probably secrete the vascular endothelial growth factor (VEGF) or other factors which attract capillaries. And this is what we can observe, that the capillarization is improved after injection of cardiac precursor cells.

Another possibility to improve the vascularization should be mentioned, i.e. embryonic stem cells can also develop endothelial cells and therefore together with the cardiac precursors we could inject precursors for endothelial cells. Hence for future therapies there are several ideas to restore a functional myocardium which is vascularized.

Mr T. Treasure (London, UK): A great talk, enjoyed it enormously, and it is probably the most promising thing I have seen about cell therapies. It promises a great future for the patient with an infarct, but I don't see that it promises a great future for cardiac surgeons looking for work to do, because Keith Dawkins has already told us that he would like to do this through the cavity of the ventricle with his catheter. So it is great science and I welcome it but the question for us is "Is it a task for cardiac surgeons?" Probably not.

Dr Hescheler : I am in favor of the cardiac surgeon, certainly, because we are working together with cardiac surgeons (Prof. Dr. Welz from the University of Bonn). I am convinced that the cardiac surgery has some major advantages against other disciplines in respect to stem cell therapy. Most importantly there are several reports that the usage of catheter for stem cell application caused serious problems since one has to inject a high number of cells through the cathether and indeed, it was shown by a clinical reserach team from South Korea that after intracoronary infusion of hematopoietic cells there was an increase of viscosity of the blood, which leads to restenosis. So I think the direct injection into the ventricle wall is still the best way, and I guess that would be best applied by cardiac surgeons.

Dr C. Valfre (Treviso, Italy): I want to ask you if those green cells that you inject and implant, living cells, I suppose, have you proven the aerobic metabolism of them by staining?

Dr Hescheler : The metabolism?

Dr Valfre : Aerobic metabolism. It has to be aerobic because otherwise it is not cardiomyocytes. It can be a myocyte.

Dr Hescheler : We had a special study on that; we did not look on the metabolism by stainings but by measuring the features of cardiac precursor cells under different oxygen conditions. It came out that the very early cells still worked well under nearly zero oxygen conditions but with further development there occurred a strong negative chronotropic effect. Hence, if we inject early cardiac precursor cells into the ischaemic tissue we assume that they can survive because of their anaerobic metabolism. Later these cells develop to the adult type of ventricular myocytes and then they certainly will change the metabolism to an aerobic status.

Dr G. Buckberg (Los Angeles, CA, USA): One question I have relates to how the role of the cardiac surgeon can evolve in a microscopic–macroscopic marriage. During your introduction, you told us that a scaffold is needed for the cells to grow. Perhaps the role of the cardiac surgeon with cell transplantation is that we undertake the responsibility to create a cardiac scaffold by reshaping the ventricle. This will then provide the new cells with a structural basis upon which cell can differentiate. Conversely, if you put a cell into a circle form, the structure will remain a circle despite cell growth, and function adversely from that abnormal form. I don't think that cell growth will change geometry. Conversely, perhaps the role of the surgeon is to create the new geometry, and thus instigate the marriage between macroscopic microscopic contributions, so that cell's can be injected at the time we do the macroscopic procedure.

Dr Hescheler : Thank you for your very important remark, that just injection of isolated cells into the preexisting cardiac matrix may not be enough in the future. If there is for example a cardiac dilatation, then we have to change first the geometry of the ventricle, and then we need other methods to put the cultured cardiac precursor cells into the heart. I see an interesting alternative in the usage of preformed scaffolds which should be cellularized and vascularized. This biohybrid system could then be engrafted into the diseased ventricle by cardiac surgery techniques.

Dr M. Turina (Zurich, Switzerland): For this method to be effective, a substantial amount of muscle will have to be transplanted to provide mechanical help. Now, one of the basic characteristics of human myocardium is its syncitial contraction: when the heart starts contracting, the whole heart contracts immediately. How do you solve the problem of electrical contact between the transplanted myocardial cells, so the impulse is propagated among them?

Dr Hescheler : I cannot give you a complete answer since we still do not know all effects which are happening after implantation of cardiac precursor cells. What we know so far is, that it is one of the earliest events in cardiac development but also after implantation of cardiac precursor cells into the damaged myocardium, that gap junctions are formed ensuring an early electrical coupling between cells. In line with this observation we never faced problems with arrhythmias after implantation of cardiac precursor cells.

Dr T. Wahlers (Jena, Germany): I have a very simple question. You are now talking in front of, let's say, 50 of the most important centers in Europe and the U.S., and they are offering you the force in order to incorporate your techniques spreading from one cardiac surgical center into 50.

How would you foresee the perspective, the future, in interacting with the cardiac surgical community as a whole with regards to the scientific techniques you offer now?

Are they inviting you too early, 5 to 10 years too early, or if you would be able in order to foresee a trial or something else, how would you approach that problem?

Dr Hescheler : You know, for me as a more basic scientist, it is really a dream that our techniques would go to clinics, and if you look in the past, many medical techniques and especially techniques in cardiac surgery have been developed through physiology. So it is really important for me that there is a close contact between our fields, and I would like to go much faster into joint projects than we are now.

It is quite evident that the assignment of a clinical cell replacement therapy based on embryonic stem cells needs several steps of futher development: (i) one has to perfect the lineage selection methods; (ii) one has to establish standart operation procedures for differentiation protocols; (iii) one has to go to mass culture protocols and to be able to produce highly purified cardiac cells in a large scale within grams to kilograms. (iv) Of course the cardiac precursor cells have to be produced from human embryonic stem cells which are free of any contaminations. And therefore the necessary political decisions have to be taken in order to allow the generation of new human embryonic stem cells under GMP conditions in Germany.

(v) The generation of new embryonic stem cell lines is also necessary in order to prevent rejection. Here the easiest and most promising way is to establish a stem cell bank with approx. 1000 cell lines. With this we could always find an almost perfect matching of the HLA type of the patient and that of the embryonic stem cells.

Of course, we, as basic scientists, cannot do all this work alone, so we need the help of people who are more powerful in establishing those structures in particular in respect of the clinical usage. Worth mentioning that all these steps need time and there are still many problems to be solved between the proof of principal — which is done — and the first treatment of patients

Dr Wahlers : Time frame?

Dr Hescheler : Around 10 years, but it also depends on the efforts and money. If we really try our best and if we work together, then I think it could be probably done much sooner.

	Footnotes

 
Presented at the EACTS Symposium for the Future of Cardiac Surgery, Frankfurt, Germany, July 1–2, 2004.

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