CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Exploring "cardiac regeneration" with heterotopic heart transplants and co-culture experiments

The mammalian heart responds to tissue injury by scar formation and tissue remodelling and has been traditionally considered as a tissue unable to regenerate. However, during the last several years there is growing interest in exploring ways to promote cardiac regeneration using stem cells derived from other tissues or from the heart itself. Injection of bone marrow-derived cells into the infarcted myocardium has been reported to induce extensive regeneration through transdifferentiation, but this conclusion has been challenged by other studies. Transdifferentiation of circulating stem cells has into cardiomyocytes has likewise been described in human sex-mismatched heart transplants, although other studies reported only negligible contribution of host-derived cells in this setting. We have reinvestigated the destiny of coirculating cells in experimental heart transplants. In addition, we examined the role of heart interstitial cells in co-culture experiments.

 

Heart transplants. To investigate engraftment and differentiation of host-derived cells in heart transplants, we have used an experimental model of heterotopic heart transplantation in rats (Fig. 1). Normal donor rat hearts were transplanted into the abdominal cavity of GFP+ transgenic recipient rats and were analyzed 15 and 90 days after surgery (Ausoni et al, 2005). Using the GFP reporter transgene we were able to identify host-derived cells engrafting the donor heart genetically. Most transplants contained GFP+ mature cardiomyocytes (Fig. 2). However, systematic counting in the transplants showed that the proportion of GFP+ cardiomyocytes was negligible (0.0005% to 0.008% of all cardiomyocytes), thus does not significantly contribute to repopulation of the damaged myocardium. These relative proportions did not change after granulocyte-colony stimulatory factor (G-CSF) treatment, despite evidence for sustained bone-marrow cell mobilization.

 

The formation of GFP+ cardiomyocytes in the transplanted hearts could be due to recipient circulating cells migrating into the transplant and transdifferentiating into mature cardiomyocytes. Alternatively, circulating cells migrated into the graft could fuse with mature cardiomyocytes. Heterotopic heart xenografts were used to distinguish between transdifferentiation and fusion (Dedja et al, 2006). Both transgenic mice expressing nuclear beta-galactosidase under the control of the cardiac troponin I promoter and wild-type hamsters served as heart donors, while GFP+ transgenic rats were used as recipients (Fig. 1). The results of this experiment were clear: without exception, all GFP+ cardiomyocytes also expressed donor markers, therefore they must derive from cell fusion, not transdifferentiation (Fig. 3).

 

Co-culture experiments. Recent data indicate that the mammalian heart contains a pool of undifferentiated progenitor cells, present in heart interstitial tissue, which can give rise to different cell types, including cardiomyocytes. However, the nature of these cells is object of debate. We have re-examined the cell composition of the cardiac interstitium in adult rat heart with respect to i) expression of markers of cardiac commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We found that most interstitial cells, including fibroblasts and a subpopulation of pericytes, from the adult heart, but not from other tissues, express the transcription factor GATA4 (Zaglia et al, 2009) (Fig. 4 A-C). GATA4+ cardiac interstitial cells do not display myogenic potential in culture. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone 3 (H3P) and incorporating 5-bromodeoxyuridine (BrdU) in the co-cultures (Fig.4 D-F). Thus, cardiac fibroblasts are able to modulate the behaviour of adult cardiomyocytes, a property that might be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.

Publications

  • Zaglia T, Dedja A, Candiotto C, Cozzi E, Schiaffino S, Ausoni S (2009) Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes. J. Mol. Cell. Cardiol. 46:653-62.
  • Dedja A, Zaglia T, Dall'Olmo L, Chioato T, Thiene G, Fabris L, Ancona E, Schiaffino S, Ausoni S, Cozzi E (2006) Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts. FASEB J. 20:2534-6.
  • Ausoni S, Zaglia T, Dedja A, Di Lisi R, Seveso M, Ancona E, Thiene G, Cozzi E, Schiaffino S (2005) Host-derived circulating cells do not significantly contribute to cardiac regeneration in heterotopic rat heart transplants. Cardiovasc. Res. 68:394-404.

Grants

European Commission, VI Programme: HeartRepair (Integrated Project) "Heart Failure and Cardiac Repair" (4 yrs: 2006-2009). € 344.000,00

Collaborations

  • Emanuele Cozzi and Arben Dedja, Department of Medical and Surgical Sciences, University of Padova, and CORIT (Consorzio per la Ricerca sul Trapianto d'Organi), Italy.
  • Angelo Avogaro and Giampaolo Fadini, Department of Clinical and Experimental Medicine, University of Padova, Italy.

 

PI photo

Stefano Schiaffino

Contact information

email  E-mail

email  049 7923232

Participating staff

Simonetta Ausoni
Assistant Professor

Tania Zaglia
Postdoc