Abstract 9107: Pim-1 Kinase Increases Non-Random Chromatid Segregation in Cardiac Progenitor Cells

Abstract

Rationale: A cell has DNA strands of different replication ages because DNA replication is semi-conservative. Double-stranded DNA molecules possess one DNA strand that is one generation older than the other. Non-sister chromatids containing newly synthesized DNA and ‘old’ DNA strands are non-randomly partitioned between mother and daughter cells. Stem cells are presumed to non-randomly segregate chromatids to determine cell fate of daughter cells, although experimental evidence is lacking. Resident cardiac progenitor cells (CPC) present in the adult heart have been used for cell-based treatment of myocardial damage but the factors determining the stemness and lineage commitment of these cells are poorly understood. Cardio protective kinase Pim-1 increases asymmetric cell division in vivo, proliferation and commitment of CPCs after adaptive transfer in pathological injury model but its role in non-random segregation is unknown. Objective: Establish role of Pim-1 on non-random chromatid segregation and correlate with daughter cell fate determination in cardiac progenitor cells. Methods and Results: Non-random segregation was tracked by incorporation of bromodeoxyuridine (BrdU), a thymidine analogue labeling newly synthesized DNA strands. For label retention assay, cells were labeled for many generations and blocked in second cytokinesis during chase in BrdU free media. Two daughter nuclei within a cell body had different BrdU intensity. Density measurements showed that 4.6% of CPCs outside the 95% confidence interval for equal BrdU intensity between daughter nuclei indicating non-random segregation of BrdU labeled chromatids. Genetic engineering of CPCs to express Pim-1 kinase greatly enhanced non-random segregation (12.6% vs 5.6% GFP expressing cells). Non-random segregation was further confirmed by asymmetric BrdU segregation in anaphase cells from second mitotic shake off during chase. Conclusions: CPCs non-randomly segregate chromatids as demonstrated by selective segregation of BrdU labeled chromatids which is twofold increased in cells overexpressing Pim-1 kinase. Future Directions: The relationship of non-random chromatid segregation to identity of stem cells fate remains to be determined.