Overexpression of integrin-linked kinase induces cardiac stem cell expansion

Objective: Recent evidence suggests that the adult heart contains stem cells that are capable of self- renewal as well as multilineage differentiation. However, their inherent capacity for self-renewal is limiting to cell replacement applications. Integrin-linked kinase is a multifunctional protein kinase that activates Wnt target genes implicated in the symmetric replication of embryonic stem cells. Methods: Primary cultures derived from human fetal cardiac tissue (19-22 weeks' gestation) were grown in serum- free media and evaluated for the presence of cardiac progenitor cells. The effect of integrin-linked kinase was ascertained by adenoviral overexpression. Results: Cultures infected with wild-type integrin-linked kinase yielded a significant (P = .001), approximately 5-fold increase in both the absolute number and the frequency of c-Kit-positive, myosin-negative cells. Cardiospheres, comprised on morphologically homogeneous, anchorage-independent cells, were reproducibly present at days 7 to 10 and formed derivative cardiospheres in multiple passages. Integrin-linked kinase infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested, compared with untreated and virus controls (P = .001). Secondary spheres transferred to differentiation medium and 5-aza-deoxycytodine (10 mu mol/L) generated cells exhibiting biochemical evidence of differentiation into cardiomyocytes, smooth muscle cells, and endothelial cells. Conclusions: This study demonstrates that self-renewing cardiospheres generated from human fetal cardiac cells are composed of cells exhibiting the properties of stem cells, including the capacity for self-renewal and multilineage differentiation. Our results suggest that integrin-linked kinase promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine.