Myoblasts Survive Intracardiac Transfer and Divide Further after Transplantation

(#2002-11101 ... June 27, 2001)

Helmut Gulbins, MD, Sonja Schrepfer, MD, Antje Uhlig, Angelika Goldemund, Martin Oberhoffer, Hermann Reichenspurner MD, PhD, Bruno Meiser, MD, Bruno Reichart, MD

Department of Cardiac Surgery, University Hospital Grosshadern, Munich, Germany

ABSTRACT

Objective: Skeletal myoblasts have been proven to survive transplantation into myocardial scar tissue. The objective of this study was to evaluate whether these cells can also be transferred into vital myocardium and maintain their ability for cell division after transplantation. In addition, an intravital fluorescence dye for marking these cells was evaluated.

Material and Methods: Skeletal myoblasts were harvested from male Lewis rats (n = 6) and then expanded in culture. Before implantation, these cells were trypsinized and labeled using an intravital fluorescence dye (PKH-26). Syngenic myoblast transfer to recipient female Lewis rats (n = 36) was used to simulate autologous transplantation. Under general anesthesia, the rats received injections of 10⁶ myoblasts via a subxyphoidal approach into the apex of the heart. The animals were then divided into 3 groups (n = 10 each). The animals were sacrificed at several time points, and the hearts were harvested for histologic examination: group A, 7 days postoperatively; group B, 14 days postoperatively; and group C, 28 days postoperatively. An additional group, group D (n = 6), served as a control group; these animals were injected with only cell medium. Corresponding to the study groups, 2 animals of this control group were sacrificed at each time point, and the hearts were explanted. At histological examination, 8-μm sections were investigated to identify surviving stained cells. For further evaluation, the sections were stained using monoclonal antibodies against n-cam, desmin, and α- actin.

Results: No fluorescing cells were found in any hearts of rats in the control group. Surviving fluorescing myoblasts were found in 9 of 10 hearts of groups A and C and 8 of 10 hearts of group B. Labeled myoblasts were located in the intercellular spaces between the myocardial fibers. Fibrotic or inflammatory reactions could not be identified around the injection site in any hearts of the study groups. Immunohistochemical staining results showed that the labeled cells expressed n-cam, desmin, and α-actin. The myoblasts had regained their physiologic structures and had started to form myofibers. In groups B and C, more n-cam-positive cells than labeled cells were found, indicating further cell division.

Conclusions: Intravital fluorescence staining with PKH- 26 dye proved to be an easy and reliable method for identifying cells after cellular transplantation. Myoblasts survived an intracardiac transfer, regaining their physiologic structures and maintaining their ability for further cell division.

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