Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells

Kathyjo A. Jackson, Susan M. Majka, Hongyu Wang, Jennifer Pocius, Craig J. Hartley, Mark W. Majesky, Mark L. Entman, Lloyd H. Michael, Karen K. Hirschi, and Margaret A. Goodell


Introduction

The blockage of a blood vessel eventually results in myocardial ischemia, a condition that promotes rapid myocardial necrosis of the heart and scar tissue formation. Consequent re-perfusion of the vessel and heart repairs the ventricular wall to only a limited capacity. Cardiac myocytes cannot replicate, and the ventricular walls are replaced by scarring. Without treatment, ischemic heart muscle pumps blood and oxygen less efficiently, and may lead to heart failure and stroke.

This study seeks to explore the potential of adult stem cell therapy for repairing ischemic heart muscle. The researchers transplanted side population (SP) cells, enriched hematopoietic CD34^- stem cells, into mice rendered ischemic by coronary artery blockage. SP cells were shown previously to regenerate cardiac myofibers and blood vessels in injured cardiac tissue. Subsequent measurements of cardiomyocyte and endothelial cell differentiation were taken based on donor marker identification (lacZ gene expression).

Materials and Methods

Bone marrow specimens were extracted from the tibias and femurs of 6-Rosa transgenic mice, which express lacZ. To isolate SP cells, which account for 0.05% of whole bone marrow, the specimens underwent Hoechst dye staining and specific protein expression—most SP cells are positive for the markers c-Kit and PECAM-1. A triple-laser instrument was used to sort the desired SP cells through fluorescence emission analysis; the purity of SP cells in the sorted samples always exceeded 91%.

2,000 SP cells isolated from male 6-Rosa-Ly-5.2 mice were injected into irradiated female 6-Ly-5.1 mice. 10 weeks after the stem cell transplantation, the left anterior descending coronary artery of each mouse was blocked for 60 minutes, and followed by reperfusion. Two or four weeks after the injury, the hearts were removed and frozen for immunohistochemical analysis.

Frozen sections of tissue were fixed with paraformaldehyde and stained for lacZ expression. Specific antibodies were used to identify cardiac muscle (anti-α-actinin) and endothelial cells (anti-ICAM-1). Hematopoietic cells were visualized using anti-Ly-5-biotin. Slides of stained tissues were analyzed with fluorescence and differential interference contrast microscopy. SP cells and their progeny could be detected through lacZ expression, since they were derived from the 6-Rosa transgenic mice, which express lacZ.

Results

22 mice received bone marrow transplants; the coronary artery blockage procedure was performed on 19 of the mice—the remaining 3 mice acted as the control, where no procedure was done. After the hearts were sectioned, lacZ staining existed mostly in the capillaries. Co-staining with antibodies against Flt-1 and ICAM-1 illustrated that the SP cells had migrated to the injured heart through the circulation, localized to new vessels, and integrated into the surface linings (Figure 1). There were no lacZ-positive cells in the control mice.

Figure 1—Sections incorporated into vascular endothelial cells. (a-d): X-gal stained tissue for SP cell incorporation. (e-l): cardiac tissue as above, but stained for endothelial marker expression (Flt-1 and ICAM-1).

LacZ-positive cells were also incorporated into cardiac muscle; cardiac tissue from treated mice included staining throughout the heart. LacX-positive cells clustered at the edge of the myocardial scar, where most of the damage resides. Figure 2 illustrates the distribution of lacZ staining within cardiac tissue.

Figure 2—LacZ staining accumulates at the border of the myocardial scar. (a): X10 photograph of a mouse myocardial infarction after 4 weeks. The arrow points at the lacZ staining location. (b): X20 pictograph of the same lacZ area. Open arrowheads indicate macrophages. (c) X40 pictograph. (d) macrophage density after 1 hour of ischemia and 3 hours of perfusion.

Table 1 shows the array of lacZ-positive cells in various mouse subjects. 100 sections from each animal were analyzed, and the degree of lacZ staining was quantified. Each section contained about 13,000 cardiomyoctes; there was a mean engraftment of 0.02% for SD cells of all cardiomyocytes. 100 vessel profiles were analyzed per tissue section in terms of cell engraftment. The mean prevalence of lacZ-positive vessels was about 3%.

Table 1—Table illustrating the percent engraftment for cardiomyocytes and endothelial cells with regards to lacZ cell presence. Cardiac tissue analyses were conducted on 4 mice.

Discussion

Because myocardial infarction is a major cause of heart failure, new sources of cells are needed for transplantation and heart muscle regeneration. SP cells represent a progenitor population both accessible from a patient’s own tissue and adaptable for clinical use. This study demonstrates the ability of purified stem cells derived from mouse bone marrow to participate in cardiac muscle regeneration following ischemia. The SP cells marked with the lacZ gene migrated into cardiac myofibers, and aided in the regeneration process. In addition, the study showed that lacZ-positive cells could participate in vascularization within heart tissue. The utilization of SP cells into the regeneration of endothelial cells and cardiomyocytes suggests the role of circulating stem cells for tissue repair.

Critique

The study demonstrates the therapeutic potential of SP cells for cardiac tissue regeneration. Clinical trials on mice do indeed show incorporation of stem cells into the heart’s scar formation, as well as new endothelial tissue. The research does a good job in establishing a control group to see the effect of SP cells on a healthy mouse heart. Unfortunately, many limitations exist within this study. The researchers rely on lacZ expression as the major identifier of migrating SP cells. Other identifiers such as surface markers and morphology could have been obtained to establish a more reliable identification method. The study analyzed only 4 mice with regards to heart tissue, due to the large number of mice that died after the coronary artery procedure. A larger sample size may reveal better results. In addition, the amount of SP cell engraftment was relatively low for both cardiac muscle and endothelial tissue. Further studies involving SP cells and the tissue regeneration rate of live mice would be helpful.