Overexpression of Integrin-Linked Kinase Induces Cardiac Stem Cell Expansion
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Objective:Recent evidence suggests that the adult heart contains stem cells, which are capable of self-renewal as well as tissue-specific, multi-lineage differentiation. However, their inherent capacity for self-renewal is limiting to cell replacement applications. Integrin-linked kinase (ILK) is a multi-functional protein kinase, which coordinates signal transduction by integrins and growth factor receptors, and activates Wnt target genes implicated in the maintenance and symmetric replication of embryonic stem cells. We tested whether cardiac stem cell population was susceptible to amplification through ILK gain-of-function.
Methods: Fetal hearts of gestational ages 19-22 weeks were cultured to 60-70% confluency, and infected with replication defective adenovirus containing wild type (Ad.ILK), or virus control. Effective gene transfer was confirmed by more than 80% GFP positivity and by ~ 3-fold increase in ILK protein expression in cell cultures.
Results:Cultures infected with wild type ILK yielded a significant (p=0.001), ~ 5-fold increase in both the absolute number and the frequency of c-KitPOS, myosinNEG cells, which reached ~ one cell in 250. To determine if primary human fetal cardiac cells generate cardiospheres in vitro, cells were infected with Ad.ILK or control virus and plated in serum-free medium supplemented with 20ng/ml each of EGF and bFGF at clonal density of a single cell per well. Primary cardiospheres (CS), comprised on morphologically homogeneous cells, were reproducibly present at day 7-10, and formed derivative CS in multiple passages. ILK 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=0.001). Secondary spheres transferred to differentiation medium consisting of IMDM with 10% FBS and 5-Aza-deoxycytodine (10uM) 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 comprised of cells exhibiting the properties of stem cells, including the capacity for self-renewal and multilineage differentiation. Overexpression of ILK resulted in an ~10-fold increase in the frequency of sphere-initiating cells. Importantly, ILK-transformed stem cells are shown to be equally susceptible to cardiac differentiation, even while exhibiting an increased capacity for proliferation and CS formation. Our results suggest that ILK promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine.
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