Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration

Abstract

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Figure 1

In vitro cardiac differentiation of endothelial cells. (A–C) Double fluorescence of a coculture of neonatal rat cardiomyocytes, stained with anti-MyHC MF20 (red in A) with endothelial progenitors labeled with a GFP lentiviral vector (green in B). Arrows indicate a double-labeled cell (orange in the merged figure, C). (D and E) Coculture of endothelial progenitors, freshly isolated from MLC1/3F-nLacZ day 9 embryos with neonatal rat cardiomyocytes, stained with X-Gal (D) and with anti-cardiac troponin I (E). Arrows indicate a binucleated cell expressing β-galactosidase in the nucleus and cardiac troponin I in the cytoplasm. (F–H) Double fluorescence of a coculture of neonatal rat cardiomyocytes, prelabeled with 6′-carboxyfluorescein (green in G), with endothelial progenitors labeled with an adenoviral vector expressing lacZ and stained with an anti-β-galactosidase antibody (red in F). Arrows indicate one of several endothelial cells where fluorescein has entered through open junctions (orange in the merged figure, H). (K–M) Double fluorescence of a coculture of neonatal rat cardiomyocytes, stained with anti-MyHC MF20 (red in K) with HUVEC labeled with a GFP lentiviral vector (green in L). The double arrow indicates one double-labeled cell (orange in the merged figure, M), whereas the arrowhead indicates a HUVEC that has not trans-differentiated. (N) Electron micrograph showing two close cells, both containing sarcomeres (arrows), one of which is also labeled with Bluo-Gal (arrowhead), whereas the other is not (×3,000). (J) Higher magnification (×5,000) of an area of N of a cell showing Bluo-Gal labeling (arrowhead) among sarcomeres (arrow).

Figure 2

In vivo cardiac differentiation of endothelial cells. Double fluorescence of a section of normal mouse heart (A–C) and of an infarcted mouse heart (D–F), 2 weeks after injection of endothelial progenitors labeled with a GFP lentiviral vector (green in B and E). The sections have been stained with anti-MyHC MF20 (red in A and D). Nuclear staining (Hoechst) is shown in C and F. In the uninjured heart, very few double-labeled cells could be detected (arrows in A–C). In contrast, a large number of double-labeled cells were present in the infarcted heart (arrows in D–F) in the area of injection; arrowheads indicate normal myocardium, and asterisks indicate an infarcted area devoid of injected cells.

Figure 3

In vitro cardiac differentiation of different types of endothelial and nonendothelial cells. Endothelial progenitors from embryonic aorta (EEC) and from embryonic stem cells (44b), endothelial cells from adult heart (5HV) and lung (1G11), and from human umbilical vein (HUVEC), mouse fibroblasts (3T3), and neural stem cells (NSC) were infected with the GFP-expressing vector and cocultured with neonatal rat cardiomyocytes as described in Materials and Methods. Cardiac differentiation in cocultures was scored by counting double-labeled cells (GFP⁺/MyHC⁺) in 30 randomly selected fields and expressing the number obtained as a percentage of total GFP⁺ cells. Data are the average of at least three separate experiments with SE ranging within 10% of the mean. In a separate set of experiments, the same cells were exposed to BMP4 and FGF2 as detailed in Materials and Methods, and after 5 days they were stained for the expression of MyHC. No positive cells were scored in two separate experiments. Finally, in one experiment, the same molecules were added to cocultures of the same cell lines with cardiomyocytes.

Figure 4

In vitro cardiac trans-differentiation of endothelial cells. Double fluorescence of a coculture of neonatal rat cardiomyocytes with HUVEC, stained with anti-MyHC polyclonal antibody (green in A), with anti-von Willebrand factor monoclonal antibody (red in C). Nuclear staining (Hoechst) is shown in B. The arrow indicates von Willebrand factor-containing granules in the cytoplasm of a differentiated cardiomyocyte (orange in the merged figure, D).