Background Currently, there is great interest in the potential therapeutic benefit of bone marrow-derived hematopoietic stem cells (HSCs) in repopulating the injured heart. Side Population (SP) cells are an extremely small and homogenous population of promising hematopoietic cells which are isolated based on their capacity for effluxing the DNA-binding dye Hoechst 33342. SP cells have been shown to possess great functional plasticity, including the capacity to form cardiomyocytes and endothelial cells following ischemic injury.
Hypothesis Side Population hematopoietic progenitor cells migrate and contribute to the replacement of myocardial, endothelial and smooth muscle cells damaged during myocardial injury.
Methods Bone marrow expressing green fluorescent protein (GFP) was harvested from the tibias and femurs of 7 male GFP chimeric BL/6 mice and stained with Hoechst 33342. Marrow cells positive for GFP and negative for Hoechst 33342 were isolated using a FACS DiVa Vantage (Becton Dickinson, San Jose) Fluorescence Activated Cell Sorting System. In two experiments, 69 000 and 196 000 cells that excluded the dye were sorted from the marrow population. After each cell purification, these GFP+ SP samples were divided into two aliquots and injected either intravenously or directly into the myocardium of female BL/6 mice. Prior to injection, recipient female animals were intubated and given irreversible myocardial injury via permanent ligation of the left anterior descending coronary artery. These mice were kept alive until sacrifice at 28-day time-points.
Results Currently all four mice injected intracardially and intravenously with GFP positive SP cells are surviving. Analysis of the hearts of these animals will be undertaken at 28 days in late August 2003. Comparison of intravenous and intracardial injection methods will also be undertaken. SP cell differentiation and contribution to the repair, remodeling or regeneration of the ischemic injury will be analyzed by confocal microscopy.
Conclusion If successful, the results will demonstrate the first use of a challenging and powerful model of murine myocardial infarction injury at the iCAPTUR4E Centre. In addition, we will show successful isolation, introduction, and differentiation of Side Population stem cells in the damaged heart.
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