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

Abstract

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Figure 1

Gene-expression analysis of bone marrow SP cells. (a) Normal murine bone marrow is stained with Hoechst 33342. The indicated SP population comprises around 0.05% of total bone marrow cells. (b) The majority of SP cells are positive for the markers c-Kit and PECAM-1. (c) RT-PCR analysis of purified SP cells. m, marker; VE-CAD, VE-cadherin; FVIII, factor VIII.

Figure 2

Experimental schema. SP cells were purified from bone marrow (BM-SP) obtained from C57Bl/6-Rosa26 transgenic mice. These cells were transplanted into lethally irradiated recipients. After 2 months, stable engraftment was determined by establishing the presence of lacZ-positive peripheral blood cells. Highly engrafted animals were subjected to coronary artery ligation for 60 minutes, followed by reperfusion. Two to four weeks later, the surviving animals were sacrificed for analysis of lacZ incorporation into cardiac tissue.

Figure 3

Incorporation of SP cells into vascular endothelial cells. (a–d) X-gal–stained section of cardiac tissue from an infarcted SP cell-transplant recipient; b and d show magnifications of the indicated capillaries from a and c. (e–l) Cardiac tissue as above, stained for expression of endothelial markers. An enlargement of the vessel stained for lacZ in e is seen in f. The same vessel is shown in g, stained with Flt-1, and in h, with ICAM-1. An enlargement of the vessel stained for lacZ in i is seen in j. The same vessel is shown in k, stained for Flt1, and in l, for ICAM1. a, c, e, and i: ×200; b, d, f–h, and j–l: ×1,000.

Figure 4

Incorporation of SP cells into cardiomyocytes. (a) Negative control: C57Bl/6 cardiac tissue stained for lacZ expression. (b) Positive control: C57Bl/6-Rosa26 cardiac tissue stained for lacZ expression. This typical section demonstrates both patterns of punctate and whole-fiber staining. (c) Cross-section of a heart from an SP cell–transplant recipient, which received an infarct. (d) Longitudinal section of an SP cell–transplant recipient, which received an infarct. (e–h) LacZ and α-actinin costaining of lacZ-positive fibers. (i) CD45 costaining of the section in g and h. (j) Anti-CD45 staining of spleen (positive control). Sections were stained with X-gal, and LacZ-positive sections were subsequently stained for α-actinin (f, h) and CD45 (i), and the sections were photographed.

Figure 5

LacZ staining occurs primarily at the border of myocardial infarction. (a) Lower-power (×10) photograph of mouse myocardial infarction after 4 weeks. The arrowhead points to the location of lacZ staining shown in b and c. The lighter pink tissue to the left and above the arrowhead is primarily fibrotic and results from the infarction. (b) Higher-power (×20) photograph of the same section dual stained for lacZ and the antimacrophage Ab F480. The open arrowhead indicates a macrophage, the closed arrowhead indicates lacZ-positive cardiomyocytes (the same region shown in Figure 4, g and h). (c) Higher-power photograph of the same section (×40). (d) Macrophage density of a cardiac section after 1 hour of ischemia and 3 hours of reperfusion. The open arrowheads indicate two of the many macrophages present. The counterstain is eosin.