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,1
,1,¶,
,#,2,2,**,2
* Department of Medical and Surgical Sciences, University of Padova, Padova, Italy;
CORIT (Consorzio per la Ricerca sul Trapianto d’Organi), Padova, Italy;
Department of Biomedical Sciences, University of Padova, Padova, Italy;
CNR Institute of Neurosciences; Padova, Italy;
|| Institute of Pathological Anatomy, University of Padova, Padova, Italy;
¶ Clinica Chirurgica 3°, Padova General Hospital, Padova, Italy;
# Venetian Institute of Molecular Medicine, Padova, Italy;
** Direzione Sanitaria, Padova General Hospital, Padua, Italy
2Correspondence: S.S. and S.A.: Department of Biomedical Sciences, University of Padova, Via G. Colombo 3, Padova 35121, Italy. E-mail: stefano.schiaffino{at}unipd.it; ausoni{at}bio.unipd.it; E.C.: Clinical and Experimental Transplantation Immunology, Department of Medical and Surgical Sciences, University of Padova, Ospedale Giustinianeo, Via Giustiniani Padova, Italy. E-mail: emanuele.cozzi{at}unipd.it
SPECIFIC AIMS
Cardiomyocytes expressing host markers have been detected in human and rat heart allografts, thus supporting the view that circulating cells can contribute to cardiac regeneration. It has yet to be established, however, whether host-derived cardiomyocytes result from the transdifferentiation of circulating stem cells or from cell fusion. To address this issue, we have used heterotopic cardiac xenografts, in which markers of both donor and recipient cells are available.
PRINCIPAL FINDINGS
1. Heterotopic cardiac xenotransplantation as a model for investigating the contribution of circulating cells to cardiac regeneration
In the heterotopic cardiac transplantation model, a donor heart is grafted into the abdominal cavity of the host, with anastomoses between graft aorta and host’s abdominal aorta, and between graft pulmonary artery and host’s inferior vena cava. The grafted heart beats but is not loaded, so it progressively becomes atrophic. It also suffers tissue damage due to ischemia-reperfusion injury and the host’s immune response. Grafts from two xenotransplantation groups (hamster-to-rat and mouse-to-rat) and a control rat allotransplantation group were examined 2 wk after surgery. Golden Syrian hamsters or transgenic mice expressing nuclear beta-galactosidase under the control of the cardiac troponin I (cTnI) promoter served as organ donors. Recipients were GFP transgenic rats characterized by a strong, homogenous GFP expression in all tissues. Microscopic examination showed a variable degree of tissue damage in both allografts and xenografts, with infiltration of inflammatory GFP+ host cells expressing the CD-45 marker and focal scar remodeling. Heterotopic cardiac transplants thus provide an appropriate model for exploring the potential of circulating stem cells for spontaneously homing into a damaged heart and contributing to its regeneration.
2. Undifferentiated host-derived cells expressing early cardiac markers in heterotopic cardiac xenografts
A number of small undifferentiated cells labeled by the host marker GFP and staining for early cardiac markers, GATA-4 or MEF2C, were detected in the grafts. These cells were found either in the inflammatory infiltrate or in well-preserved myocardium. Markers of advanced cardiac differentiation, such as sarcomeric proteins, were never detected in these cells.
3. Hybrid cardiomyocytes derived by cell fusion in hamster-to-rat heterotopic cardiac xenografts
Seven of eight transplanted hamster hearts revealed occasional GFP+ cells with the typical size, morphology, and cross-striation of mature cardiomyocytes and staining for sarcomeric myosin heavy chain (MyHC) and cTnI (Fig. 1
A–D). These cells were mainly distributed within well-preserved myocardium, close to areas of tissue damage. In hamster hearts sectioned from the apex to the base, systematic GFP+ cardiomyocyte counts showed values ranging from 0.0001% to 0.0344%, comparable with those of the control allotransplantation group. We used antihamster specific antisera (obtained from nonimmunosuppressed rats bearing hamster heart grafts) to determine whether these cells derive from the fusion or transdifferentiation of circulating cells. After absorption on rat tissue to remove cross-reactivity with rat antigens, the antisera produced a bright stain at the surface membrane level in the hamster heart but no stain in the rat heart (Fig. 1E-H
). When applied to sections of the xenografts, the antihamster antisera strongly stained all GFP+ cardiomyocytes, without exception (Fig. 1F, I
).
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4. Hybrid cardiomyocytes derived by cell fusion in mouse-to-rat heterotopic cardiac xenografts
In a second experimental model, adult cTnI/lacZ transgenic mice, expressing the nuclear-targeted lacZ marker exclusively in cardiomyocytes, were used as organ donors (Fig. 2
). Very rare GFP+ cardiomyocytes were identified in the mouse-to-rat xenografts; all of them coexpressed the GFP host marker and the nuclear beta-galactosidase donor marker.
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CONCLUSIONS AND SIGNIFICANCE
Two different cardiac xenotransplantation models (Fig. 3
) (hamster-to-rat and mouse-to-rat) were used to explore the potential of circulating cells for contributing to cardiac regeneration. The unambiguous outcome of these studies is that all graft cardiomyocytes expressing the GFP host cell marker also express donor markers. This shows that circulating cells are not transdifferentiated into cardiac muscle cells, they fuse with resident cardiomyocytes in the transplants. The cell fusion had already been demonstrated in other experimental settings but not in the transplant model.
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A second conclusion of this work is that the contribution of circulating cells to cardiac regeneration is negligible, much lower than reported in some studies on the human heart. Cell fusion events are too rare to achieve any significant clinical benefit unless a selective pressure is applied. It would therefore be important to devise procedures for enhancing the fusion process so as to exploit its therapeutic potential. The heterotopic cardiac transplantation model may offer some advantages in this respect. The explanted heart can be kept for several hours in cardioplegic solution before its reimplantation. During this time, it may be feasible to apply specific treatments, in order to identify factors able either to enhance extracardiac cell recruitment and cell fusion, or to promote the progression of transdifferentiation events, or to activate dormant endogenous cardiac stem cells.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6586fje
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